![]() RNA
integrity -- latest publications (5)
RNA integrity (1) RNA integrity (2) RNA integrity (3) RNA integrity & DNA integrity (4) RNA secondary structure (6) New RNA integrity publication: RNA-Seq -- impact of RNA degradation on transcript quantification. Gallego Romero I, Pai AA, Tung J, Gilad Y BMC Biol. 2014 12: 42 BACKGROUND: The use of low
quality RNA samples in whole-genome gene expression profiling remains
controversial. It is unclear if transcript degradation in low quality
RNA samples occurs uniformly, in which case the effects of degradation
can be corrected via data normalization, or whether different
transcripts are degraded at different rates, potentially biasing
measurements of expression levels. This concern has rendered the use of
low quality RNA samples in whole-genome expression profiling
problematic. Yet, low quality samples (for example, samples collected
in the course of fieldwork) are at times the sole means of addressing
specific questions.
RESULTS: We sought to quantify
the impact of variation in RNA quality on estimates of gene expression
levels based on RNA-seq data. To do so, we collected expression data
from tissue samples that were allowed to decay for varying amounts of
time prior to RNA extraction. The RNA samples we collected spanned the
entire range of RNA Integrity Number (RIN) values (a metric commonly
used to assess RNA quality). We observed widespread effects of RNA
quality on measurements of gene expression levels, as well as a slight
but significant loss of library complexity in more degraded samples.
CONCLUSIONS: While standard
normalizations failed to account for the effects of degradation, we
found that by explicitly controlling for the effects of RIN using a
linear model framework we can correct for the majority of these
effects. We conclude that in instances in which RIN and the effect of
interest are not associated, this approach can help recover
biologically meaningful signals in data from degraded RNA samples.
Long term storage of dry versus frozen RNA for next generation molecular studies. Seelenfreund E, Robinson WA, Amato CM, Tan AC, Kim J, Robinson SE PLoS One. 2014 9(11): e111827 -- eCollection 2014 The standard method for the storage and preservation of RNA has been at ultra-low temperatures. However, reliance on liquid nitrogen and freezers for storage of RNA has multiple downsides. Recently new techniques have been developed for storing RNA at room temperature utilizing desiccation and are reported to be an effective alternative for preserving RNA integrity. In this study we compared frozen RNA samples stored for up to one year to those which had been desiccated using RNAstable (Biomatrica, Inc., San Diego, CA) and stored at room temperature. RNA samples were placed in aliquots and stored after desiccation or frozen (at -80°C), and were analyzed for RNA Integrity Number (RIN), and by qPCR, and RNA sequencing. Our study shows that RNAstable is able to preserve desiccated RNA samples at room temperature for up to one year, and that RNA preserved by desiccation is comparable to cryopreserved RNA for downstream analyses including real-time-PCR and RNA sequencing. Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples. Zhang H, Korenková V, Sjöback R, Švec D, Björkman J, Kruhøffer M, Verderio P, Pizzamiglio S, Ciniselli CM, Wyrich R, Oelmueller U, Kubista M, Lindahl T, Lönneborg A, Rian E PLoS One. 2014 Nov 4;9(11): e111644 -- eCollection 2014 There is an increasing
need for proper quality control tools in the
pre-analytical phase of the molecular diagnostic workflow. The aim of
the present study was to identify biomarkers for monitoring
pre-analytical mRNA quality variations in two different types of blood
collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes
(PAXgene tubes). These tubes are extensively used both in the
diagnostic setting as well as for research biobank samples. Blood
specimens collected in the two different blood collection tubes were
stored for varying times at different temperatures, and microarray
analysis was performed on resultant extracted RNA. A large set of
potential mRNA quality biomarkers for monitoring post-phlebotomy gene
expression changes and mRNA degradation in blood was identified. qPCR
assays for the potential biomarkers and a set of relevant reference
genes were generated and used to pre-validate a sub-set of the selected
biomarkers. The assay precision of the potential qPCR based biomarkers
was determined, and a final validation of the selected quality
biomarkers using the developed qPCR assays and blood samples from 60
healthy additional subjects was performed. In total, four mRNA quality
biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated.
We suggest here the use of these blood mRNA quality biomarkers for
validating an experimental pre-analytical workflow. These biomarkers
were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program
which demonstrated that these biomarkers can be used as quality control
tools for mRNA analyses from blood samples.
A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity. Adamski MG, Gumann P, Baird AE PLoS One. 2014 9(8): e103917 -- eCollection 2014 Over the past decade
rapid advances have occurred in the understanding of RNA expression and
its regulation. Quantitative polymerase chain reactions (qPCR) have
become the gold standard for quantifying gene expression. Microfluidic
next generation, high throughput qPCR now permits the detection of
transcript copy number in thousands of reactions simultaneously,
dramatically increasing the sensitivity over standard qPCR. Here we
present a gene expression analysis method applicable to both standard
polymerase chain reactions (qPCR) and high throughput qPCR. This
technique is adjusted to the input sample quantity (e.g., the number of
cells) and is independent of control gene expression. It is
efficiency-corrected and with the use of a universal reference sample
(commercial complementary DNA (cDNA)) permits the normalization of
results between different batches and between different
instruments--regardless of potential differences in transcript
amplification efficiency. Modifications of the input quantity method
include (1) the achievement of absolute quantification and (2) a
non-efficiency corrected analysis. When compared to other commonly used
algorithms the input quantity method proved to be valid. This method is
of particular value for clinical studies of whole blood and circulating
leukocytes where cell counts are readily available.
Re-use of commercial microfluidics chips for DNA, RNA, and protein electrophoresis. Nguyen T, Kwak S, Karpowicz SJ Biotechniques. 2014 57(5): 267-271 Microfluidics chip
technology is a powerful and convenient alternative to agarose gels and
PAGE, but costs can be high due to certain chips being non-reusable.
Here we describe a method to regenerate, re-use, and store Agilent DNA,
RNA, and protein electrophoresis chips designed for use in the
Bioanalyzer 2100. By washing the sample wells and displacing the old
gel matrix with new gel-dye mix, we have run samples on the same chip
up to ten times with negligible loss of signal quality. Chips whose
wells were loaded with buffer or water were stored successfully for one
week before re-use.
Profiling of RNA degradation for estimation of post morterm interval. Sampaio-Silva F, Magalhães T, Carvalho F, Dinis-Oliveira RJ, Silvestre R PLoS One. 2013 8(2): e56507 An estimation of the
post mortem interval (PMI) is frequently touted as the Holy Grail of
forensic pathology. During the first hours after death, PMI estimation
is dependent on the rate of physical observable modifications including
algor, rigor and livor mortis. However, these assessment methods are
still largely unreliable and inaccurate. Alternatively, RNA has been
put forward as a valuable tool in forensic pathology, namely to
identify body fluids, estimate the age of biological stains and to
study the mechanism of death. Nevertheless, the attempts to find
correlation between RNA degradation and PMI have been unsuccessful. The
aim of this study was to characterize the RNA degradation in different
post mortem tissues in order to develop a mathematical model that can
be used as coadjuvant method for a more accurate PMI determination. For
this purpose, we performed an eleven-hour kinetic analysis of total
extracted RNA from murine's visceral and muscle tissues. The
degradation profile of total RNA and the expression levels of several
reference genes were analyzed by quantitative real-time PCR. A
quantitative analysis of normalized transcript levels on the former
tissues allowed the identification of four quadriceps muscle genes
(Actb, Gapdh, Ppia and Srp72) that were found to significantly
correlate with PMI. These results allowed us to develop a mathematical
model with predictive value for estimation of the PMI (confidence
interval of ±51 minutes at 95%) that can become an important
complementary tool for traditional methods.
![]() A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples. Sellin Jeffries MK, Kiss AJ, Smith AW, Oris JT BMC Biotechnol. 2014 14: 94 BACKGROUND: This study
compared the performance of five commercially available kits in
extracting total RNA from small eukaryotic tissue samples (<15 mg).
Total RNA was isolated from fathead minnow (Pimephales promelas)
tissues (spleen, blood, kidney, embryo, and larvae) using the Qiagen
RNeasy® Plus Mini, Qiagen RNeasy® Plus Universal, Promega
Maxwell® 16 LEV simplyRNA, Ambion MagMAX™-96 and Promega SimplyRNA
HT kits. Kit performance was evaluated via measures of RNA quantity
(e.g., total RNA amount) and quality (e.g., ratio of absorbance at 260
and 280 nm, RNA integrity number (RIN), presence of gDNA).
RESULTS: With the exception of
embryos, each kit generally extracted ≥5 μg of total RNA from each
sample. With regard to RNA quality, the RINs of RNA samples isolated
via the Plus Mini and Maxwell® 16 kits were consistently higher
than those of samples extracted via the remaining three kits and for
all tissues, these kits produced intact RNA with average RIN values ≥7.
The Plus Universal and SimplyRNA HT kits produced moderately degraded
(RIN values <7, but ≥5), while the RNA recovered via the MagMAX™ kit
tended to exhibit a high degree of degradation (RIN values <5).
CONCLUSIONS: Each kit was
generally capable of extracting the amount of RNA required for most
downstream gene expression applications suggesting that RNA yield is
unlikely to be a limiting factor for any of the kits evaluated.
However, differences in the quality of RNA extracted via each of the
kits indicate that these kits may differ in their ability to yield RNA
acceptable for some applications. Overall, the findings of this study
demonstrate that there are practical differences between commercially
available RNA extraction kits that should be taken into account when
selecting extraction methods to be used for isolating RNA designated
for gene expression analysis.
Degradation of intestinal mRNA -- a matter of treatment. Heumüller-Klug S, Sticht C, Kaiser K, Wink E, Hagl C, Wessel L, Schäfer KH World J Gastroenterol. 2015 21(12): 3499-3508 AIM: To characterize the
influence of location, species and treatment upon RNA degradation in
tissue samples from the gastrointestinal tract.
METHODS: The intestinal
samples were stored in different medium for different times under
varying conditions: different species (human and rat), varying
temperature (storage on crushed ice or room temperature), time point of
dissection of the submucous-mucous layer from the smooth muscle (before
or after storage), different rinsing methods (rinsing with Medium, PBS,
RNALater or without rinsing at all) and different regions of the gut
(proximal and distal small intestine, caecum, colon and rectum). The
total RNA from different parts of the gut (rat: proximal and distal
small intestine, caecum, colon and rectum, human: colon and rectum) and
individual gut layers (muscle and submucosal/mucosal) was extracted.
The quality of the RNA was assessed by micro capillary electrophoresis.
The RNA quality was expressed by the RNA integrity number which is
calculated from the relative height and area of the 18 S and 28 S RNA
peaks. From rat distal small intestine qPCR was performed for neuronal
and glial markers.
RESULTS: RNA obtained from
smooth muscle tissue is much longer stable than those from
submucosal/mucosal tissue. At RT muscle RNA degrades after one day, on
ice it is stable at least three days. Cleaning and separation of gut
layers before storage and use of RNALater, maintains the stability of
muscle RNA at RT for much longer periods. Different parts of the gut
show varying degradation periods. RNA obtained from the
submucosal/mucosal layer always showed a much worse amplification rate
than RNA from muscle tissue. In general RNA harvested from rat tissue,
either smooth muscle layer or submucosal/mucosal layer is much longer
stable than RNA from human gut tissue, and RNA obtained from smooth
muscle tissue shows an increased stability compared to RNA from
submucosal/mucosal tissue. At RT muscle RNA degrades after one day,
while the stability on ice lasts at least three days. Cleaning and
separation of gut layers before storage and use of RNALater, maintains
the stability of muscle RNA at RT for much longer periods. Different
parts of the gut show varying degradation periods. The RNA from muscle
and submucosal/mucosal tissue of the proximal small intestine degrades
much faster than the RNA of distal small intestine, caecum or colon
with rectum. RNA obtained from the submucosal/mucosal layer always
showed a much more reduced amplification rate than RNA from muscle
tissue [ß-Tubulin III for muscle quantification cycle (Cp): 22.07
± 0.25, for ß-Tubulin III submucosal/mucosal Cp: 27.42
± 0.19].
CONCLUSION: Degradation of
intestinal mRNA depends on preparation and storage conditions of the
tissue. Cooling, rinsing and separating of intestinal tissue reduce the
degradation of mRNA.
MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients. Kakimoto Y, Kamiguchi H, Ochiai E, Satoh F, Osawa M PLoS One. 2015 10(6): e0129338 MicroRNAs (miRNAs) are
very short (18-24 nucleotides) nucleic acids that are expressed in a
number of biological tissues and have been shown to be more resistant
to extreme temperatures and pH compared to longer RNA molecules, like
mRNAs. As miRNAs contribute to diverse biological process and respond
to various kinds of cellular stress, their utility as diagnostic
biomarkers and/or therapeutic targets has recently been explored. Here,
we have evaluated the usefulness of miRNA quantification during
postmortem examination of cardiac tissue from acute myocardial
infarction (AMI) patients. Cardiac tissue was collected within one week
of the patient's death and either frozen (19 samples) or fixed in
formalin for up to three years (36 samples). RNA integrity was
evaluated with an electropherogram, and it appears that longer RNAs are
fragmented after death in the long-term fixed samples. Quantitative PCR
was also performed for seven miRNAs and three other small RNAs in order
to determine the appropriate controls for our postmortem analysis. Our
data indicate that miR-191 and miR-26b are more suitable than the other
types of small RNA molecules as they are stably detected after death
and long-term fixation. Further, we also applied our quantitation
method, using these endogenous controls, to evaluate the expression of
three previously identified miRNA biomarkers, miR-1, miR-208b, and
miR-499a, in formalin-fixed tissues from AMI patients. Although miR-1
and miR-208b decreased (1.4-fold) and increased (1.2-fold),
respectively, in the AMI samples compared to the controls, the
significance of these changes was limited by our sample size. In
contrast, the relative level of miR-499a was significantly decreased in
the AMI samples (2.1-fold). This study highlights the stability of
miRNAs after death and long-term fixation, validating their use as
reliable biomarkers for AMI during postmortem examination.
In vitro application of ribonucleases:
comparison of the effects on mRNA and miRNA stability.Aryani A and Denecke B BMC Res Notes. 2015 8(1): 164 BACKGROUND: MicroRNA has
become important in a wide range of research interests. Due to the
increasing number of known microRNAs, these molecules are likely to be
increasingly seen as a new class of biomarkers. This is driven by the
fact that microRNAs are relatively stable when circulating in the
plasma. Despite extensive analysis of mechanisms involved in microRNA
processing, relatively little is known about the in vitro decay of
microRNAs under defined conditions or about the relative stabilities of
mRNAs and microRNAs.
METHODS: In this in vitro
study, equal amounts of total RNA of identical RNA pools were treated
with different ribonucleases under defined conditions. Degradation of
total RNA was assessed using microfluidic analysis mainly based on
ribosomal RNA. To evaluate the influence of the specific RNases on the
different classes of RNA (ribosomal RNA, mRNA, miRNA) ribosomal RNA as
well as a pattern of specific mRNAs and miRNAs was quantified using
RT-qPCR assays. By comparison to the untreated control sample the
ribonuclease-specific degradation grade depending on the RNA class was
determined.
RESULTS: In the present in
vitro study we have investigated the stabilities of mRNA and microRNA
with respect to the influence of ribonucleases used in laboratory
practice. Total RNA was treated with specific ribonucleases and the
decay of different kinds of RNA was analysed by RT-qPCR and
miniaturized gel electrophoresis. In addition, we have examined whether
the integrity observed for ribosomal RNA is applicable to microRNA and
mRNA. Depending on the kind of ribonuclease used, our results
demonstrated a higher stability of microRNA relative to mRNA and a
limitation of the relevance of ribosomal RNA integrity to the integrity
of other RNA groups.
CONCLUSION: Our results
suggest that the degradation status of ribosomal RNA is not always
applicable to mRNA and microRNA. In fact, the stabilities of these RNA
classes to exposure to ribonucleases are independent from each other,
with microRNA being more stable than mRNA. The relative stability of
microRNAs supports their potential and further development as
biomarkers in a range of applications.
Purification of high-quality RNA from
synthetic polyethylene glycol-based hydrogels.Gasparian A, Daneshian L, Ji H, Jabbari E, Shtutman M Anal Biochem. 2015 484: 1-3 Polyethylene glycol
(PEG)-based hydrogels, with variable stiffness, are widely used in
tissue engineering to investigate substrate stiffness effects on cell
properties. Transcriptome analysis is a critical method for
understanding cell physiology. However, significant RNA degradation was
observed during the process of isolating and purifying RNA from cells
encapsulated in the PEG hydrogel, thereby precluding purification of
high-quality RNA. Here, we describe a simple protocol that prevents RNA
degradation and improves the quality and yield of RNA isolated from
cells cultured in PEG hydrogels. This modification produces
high-quality total RNA suitable for RNA sequencing and microarray
analysis.
Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation Eikmans M, Rekers NV, Anholts JD, Heidt S, Claas FH. Blood. 2013 Mar 14;121(11): e81-89 Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material. Quality assessment and data handling methods for Affymetrix Gene 1.0 ST arrays with variable RNA integrity Viljoen KS, Blackburn JM. BMC Genomics. 2013 Jan 16;14:14 BACKGROUND: RNA and microarray quality assessment form an integral part of gene expression analysis and, although methods such as the RNA integrity number (RIN) algorithm reliably asses RNA integrity, the relevance of RNA integrity in gene expression analysis as well as analysis methods to accommodate the possible effects of degradation requires further investigation. We investigated the relationship between RNA integrity and array quality on the commonly used Affymetrix Gene 1.0 ST array platform using reliable within-array and between-array quality assessment measures. The possibility of a transcript specific bias in the apparent effect of RNA degradation on the measured gene expression signal was evaluated after either excluding quality-flagged arrays or compensation for RNA degradation at different steps in the analysis. RESULTS: Using probe-level and inter-array quality metrics to assess 34 Gene 1.0 ST array datasets derived from historical, paired tumour and normal primary colorectal cancer samples, 7 arrays (20.6%), with a mean sample RIN of 3.2 (SD = 0.42), were flagged during array quality assessment while 10 arrays from samples with RINs < 7 passed quality assessment, including one sample with a RIN < 3. We detected a transcript length bias in RNA degradation in only 5.8% of annotated transcript clusters (p-value 0.05, FC ≥ |2|), with longer and shorter than average transcripts under- and overrepresented in quality-flagged samples respectively. Applying compensatory measures for RNA degradation performed at least as well as excluding quality-flagged arrays, as judged by hierarchical clustering, gene expression analysis and Ingenuity Pathway Analysis; importantly, use of these compensatory measures had the significant benefit of enabling lower quality array data from irreplaceable clinical samples to be retained in downstream analyses. CONCLUSIONS: Here, we demonstrate an effective array-quality assessment strategy, which will allow the user to recognize lower quality arrays that can be included in the analysis once appropriate measures are applied to account for known or unknown sources of variation, such as array quality- and batch- effects, by implementing ComBat or Surrogate Variable Analysis. This approach of quality control and analysis will be especially useful for clinical samples with variable and low RNA qualities, with RIN scores ≥ 2. Assessing an Improved Protocol for Plasma microRNA Extraction Moret I1, Sánchez-Izquierdo D2, Iborra M3, Tortosa L1, Navarro-Puche A4, Nos P3, Cervera J5, Beltrán B3. PLoS One. 2013 Dec 23;8(12): e82753 The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen) rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer) for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice. Strategies for Biochemical and Pathologic Quality Assurance in a Large Multi-Institutional Biorepository; The Experience of the PROCURE Quebec Prostate Cancer Biobank Brimo F, Aprikian A, Latour M, Têtu B, Doueik A, Scarlata E, Hamel L, McKercher G, Saad F, Lacombe L, Carmel M, Chevalier S. Biopreserv Biobank. 2013 Oct;11(5): 285-290. Well-characterized, high-quality fresh-frozen prostate tissue is required for prostate cancer research. As part of the PROCURE Prostate Cancer Biobank launched in 2007, four University Hospitals in Quebec joined to bank fresh frozen prostate tissues from radical prostatectomies (RP). As the biobank progressed towards allocation, the nature and quality of the tissues were determined. RP tissues were collected by standardized alternate mirror-image or biopsy-based targeted methods, and frozen for banking. Clinical/pathological parameters were captured. For quality control, two presumed benign and two presumed cancerous frozen, biobanked tissue blocks per case (10/site) were randomly selected during the five years of collection. In a consensus meeting, 4 pathologists blindly evaluated slides (n=160) and graded quality, Gleason score (GS), and size of cancer foci. The quality of tissue RNA (37/40 cases) was assessed using the RNA Integrity Number. The biobank included 1819 patients of mean age: 62.1 years; serum PSA: 8ng/ml; prostate weight: 47.8 g; GS: 7; and pathological stage: T2 in 64.5%, T3A in 25.5% and T3B in 10% of cases. Of the 157 evaluable slides, 79 and 78 had benign and cancer tissue, respectively. GS for the 37 cancer-positive cases were: 6 in 9, 7 in 18 and >7 in 10 and, in most instances, in concordance with final GS. In 40% of slides containing cancer, foci occupied ≥50% of block surface and 42% had a diameter ≥1 cm. Tissue was well preserved and consistently yielded RNA of very good quality with RNA Integrity Number (RIN) >7 for 97% of cases (mean=8.7±0.7) during the five-year collection period. This study confirms the high quality of randomly selected benign and cancerous fresh-frozen prostate tissues of the PROCURE Quebec Prostate Cancer Biobank. These results strengthen the uniqueness of this large prospective resource for prostate cancer research. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples Mee BC, Carroll P, Donatello S, Connolly E, Griffin M, Dunne B, Burke L, Flavin R, Rizkalla H, Ryan C, Hayes B, D'Adhemar C, Banville N, Faheem N, Muldoon C, Gaffney EF. Biopreserv Biobank. 2011 Dec;9(4):389-398. The Saint James's Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep(®) (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy(®) and QIAamp(®) (both Qiagen), and (4) to examine the effectiveness of Allprotect(®) (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software(®)). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A(260/280) values using QIAamp were 33.2 ng/μL and 1.86, respectively, and using AllPrep were 23.2 ng/μL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng/μL and 8.16, respectively, and with AllPrep were 74.8 ng/μL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates. Veterinary and human biobanking practices: enhancing molecular sample integrity Hostetter G, Collins E, Varlan P, Edewaard E, Harbach PR, Hudson EA, Feenstra KJ, Turner LM, Berghuis BD, Resau JH, Jewell SD. Vet Pathol. 2014 Jan;51(1):270-280 Animal models have historically informed veterinary and human pathophysiology. Next-generation genomic sequencing and molecular analyses using analytes derived from tissue require integrative approaches to determine macroanalyte integrity as well as morphology for imaging algorithms that can extend translational applications. The field of biospecimen science and biobanking will play critical roles in tissue sample collection and processing to ensure the integrity of macromolecules, aid experimental design, and provide more accurate and reproducible downstream genomic data. Herein, we employ animal experiments to combine protein expression analysis by microscopy with RNA integrity number and quantitative measures of morphologic changes of autolysis. These analyses can be used to predict the effect of preanalytic variables and provide the basis for standardized methods in tissue sample collection and processing. We also discuss the application of digital imaging with quantitative RNA and tissue-based protein measurements to show that genomic methods augment traditional in vivo imaging to support biospecimen science. To make these observations, we have established a time course experiment of murine kidney tissues that predicts conventional measures of RNA integrity by RIN analysis and provides reliable and accurate measures of biospecimen integrity and fitness, in particular for time points less than 3 hours post-tissue resection. An alternate method for DNA and RNA extraction from clotted blood Zakaria Z, Umi SH, Mokhtar SS, Mokhtar U, Zaiharina MZ, Aziz AT, Hoh BP. Genet Mol Res. 2013 Feb 4;12(1): 302-311 We developed an alternative method to extract DNA and RNA from clotted blood for genomic and molecular investigations. A combination of the TRIzol method and the QIAamp spin column were used to extract RNA from frozen clotted blood. Clotted blood was sonicated and then the QIAamp DNA Blood Mini Kit was used for DNA extraction. Extracted DNA and RNA were adequate for gene expression analysis and copy number variation (CNV) genotyping, respectively. The purity of the extracted RNA and DNA was in the range of 1.8-2.0, determined by absorbance ratios of A(260):A(280). Good DNA and RNA integrity were confirmed using gel electrophoresis and automated electrophoresis. The extracted DNA was suitable for qPCR and microarrays for CNV genotyping, while the extracted RNA was adequate for gene analysis using RT-qPCR. Assessment of RNA integrity in the postmortem pig colonic tissue ex vivo Bahar B, O'Doherty JV, Sweeney T. J Anim Sci. 2012 Dec;90 Suppl 4: 22-24 Surgical removal of porcine intestinal tissue followed by an ex vivo challenge is an alternative technique of testing the anti-inflammatory effect of bioactive compounds in the intestine of live pigs. We investigated the effects of ex vivo incubation of porcine colonic tissue on the quantity and quality of total RNA over a 12-h time period. Colonic tissue of pig (n = 6) was surgically removed immediately postslaughter and incubated for 0, 3, 6, and 12 h in a humidified cell culture incubator with 5% CO(2) at 37°C. Tissue samples were processed for RNA extraction. The quantity and quality of total RNA were assessed on a NanoDrop Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. Ex vivo incubation had an effect on both the quantity (P < 0.001) and quality (P < 0.001) of total RNA. Relative to the RNA yield at 0 h (505.0 ± 48.64 μg/mg), the yield was significantly reduced after 6 h (227.6 ± 25.52 μg/mg; P < 0.001) and 12 h (159.3 ± 24.19 μg/mg; P < 0.001) of incubation. The 28S and 18S rRNA bands were visibly intact after 0, 3, and 6 h of incubation. However, after 12 h of incubation, a degraded RNA profile was evident. The RNA integrity number (RIN) values for the 0, 3, 6, and 12 h of incubation were 9.4 ± 0.10, 9.0 ± 0.10, 6.7 ± 0.17 (P < 0.001), and 3.3 ± 0.24 (P < 0.001), respectively. The transcript abundances of 4 constitutively expressed genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), beta 2-microglobulin (B2M), and peptidylprolyl isomerase A (PPIA) were reduced at both 6 and 12 h of incubation. It is concluded that ex vivo incubation of porcine colonic tissue up to 3 h postmortem generates good quality total RNA suitable for gene expression studies. RNA "traffic lights": an analytical tool to monitor siRNA integrity Holzhauser C, Liebl R, Goepferich A, Wagenknecht HA, Breunig M. ACS Chem Biol. 2013 May 17;8(5): 890-894 The combination of thiazole orange and thiazole red as an internal energy transfer-based fluorophore pair in oligonucleotides provides an outstanding analytical tool to follow DNA/RNA hybridization through a distinct fluorescence color change from red to green. Herein, we demonstrate that this concept can be applied to small interfering RNA (siRNA) to monitor RNA integrity in living cells in real time with a remarkable dynamic range and excellent contrast ratios in cellular media. Furthermore, we show that our siRNA-sensors still possess their gene silencing function toward the knockdown of enhanced green fluorescent protein in CHO-K1 cells. ![]() RNA quality assessment: a view from plant qPCR studies Die JV, Román B. Mejora Genética Vegetal, Instituto de Agricultura Sostenible, CSIC, 14080 Córdoba, Spain. J Exp Bot. 2012 Oct 8. Reverse
transcription-quantitative real-time polymerase chain reaction
(RT-qPCR) is probably the most common molecular technique used in
transcriptome analyses today. The simplicity of the technology and
associated protocols that generate results without the need to
understand the underlying principles has made RT-qPCR the method of
choice for RNA quantification. Rather than the 'gold standard
technology' often used to describe it, the performance of RT-qPCR
suffers from considerable pitfalls during general workflow. The
inconsistency of conventional methods for the evaluation of RNA quality
and its influence on qPCR performance as well as stability of reference
genes is summarized and discussed here.
Quantification of RNA integrity and its use for measurement of transcript number Brisco MJ, Morley AA. Department of Haematology and Genetic Pathology, Flinders University and Medical Centre, Bedford Park, South Australia, Australia 5042 Nucleic Acids Res. 2012 Oct 1;40(18):e144 RNA degradation can distort or prevent measurement of RNA transcripts. A mathematical model for degradation was constructed, based on random RNA damage and exponential polymerase chain reaction (PCR) amplification. Degradation, measured as the number of lesions/base, can be quantified by amplifying several sequences of a reference gene, calculating the regression of C(t) on amplicon length and determining the slope. Reverse transcriptase-quantitative PCR (RT-qPCR) data can then be corrected for degradation using lesions/base, amplicon length(s) and the relevant equation obtained from the model. Several predictions of the model were confirmed experimentally; degradation in a sample quantified using the model correlated with degradation quantified using an additional control sample and the ΔΔCt method and application of the model corrected erroneous results for relative quantification resulting from degradation and differences in amplicon length. Compared with RIN, the method was quantitative, simpler, more sensitive and spanned a wider range of RNA damage. The method can use either random or specifically primed complementary DNA and it enables relative and absolute quantification of RNA to be corrected for degradation. The model and method should be applicable to many situations in which RNA is quantified, including quantification of RNA by methods other than nucleic acid amplification. Quality Assessment of Biobanked Nucleic Acid Extracts for Downstream Molecular Analysis Karin Wahlberg, Jim Huggett, Rebecca Sanders, Alexandra S. Whale, Claire Bushell, Ramnath Elaswarapu, Daniel J. Scott, Carole A. Foy BIOPRESERVATION AND BIOBANKING Volume10, Number 3, 2012 Sample quality is of
major importance when conducting molecular analysis of nucleic acids,
and factors such as degradation, presence of impurities, and enzymatic
inhibitors may have a significant impact on the quality of data. Issues
of quality assessment become more important as the increased use of
biobanking means that whole blood samples are being stored for longer
periods. A range of commercially available kits/methods have been
specifically developed for extraction of high quality nucleic acids
from a variety of clinical samples, including blood, but there is
limited information on how best to quality assess the extracts to
determine their fitness for purpose in specific downstream
applications. In this study, we have performed nucleic acid extractions
from stored blood using a number of different methods. The resulting
extracts were analyzed by a panel of quantity and quality metrics
including UV spectrophotometry, PicoGreen fluorescence,
electrophoresis, and a PCR approach. To evaluate the relevance of
different metrics, DNA samples were subsequently assessed for their
performance in real time PCR and microarray experiments. Our findings
demonstrate that the most suitable extraction technique and quality
assessment approach depends on the required downstream analytical
method.
Establishment of a protocol for large-scale gene expression analyses of laser capture microdissected bladder tissue Horstmann M, Foerster B, Brader N, John H, Maake C. Department of Urology, Kantonsspital Winterthur, Brauerstr. 15, 8401, Winterthur, Switzerland World J Urol. 2012 May 26. PURPOSE: Lower urinary
tract symptoms (LUTS) can be caused by structural and functional
changes in different compartments of the bladder. To enable extensive
investigations of individual regions even in small bladder biopsies, we
established a combination protocol consisting of three molecular
techniques: laser capture microdissection microscopy (LCM), RNA
preamplification and quantitative polymerase chain reaction (qPCR).
METHODS: Urinary
bladders of ten mice were resected and frozen immediately or after a
delay of 15 min. Cryosections were obtained and smooth muscle was
isolated using the LCM technique. Then, RNA was extracted, including
protocols with and without DNase digestion as well as with and without
the addition of carrier RNA. Extracted RNA was either used for reverse
transcriptase (RT)-PCR plus qPCR or for a combination of RNA
preamplification and qPCR.
RESULTS: Our data showed
that with RNA preamplification, 10 μg cDNA can be regularly generated
from 2.5 ng RNA. Depending on expression levels, this is sufficient for
hundreds of pPCR reactions. The efficiency of preamplification,
however, was gene-dependent. DNase digestion before preamplification
lead to lower threshold cycles in qPCR. The use of partly degraded RNA
for RNA preamplification did not change the results of the following
qPCR.
CONCLUSIONS: RNA
preamplification strongly enlarges the spectrum of genes to be analyzed
in distinct bladder compartments by qPCR. It is an easy and reliable
method that can be realized with standard laboratory equipment. Our
protocol may lead in near future to a better understanding of the
pathomechanisms in LUTS.
Importance of RNA isolation methods for analysis of exosomal RNA: evaluation of different methods Eldh M, Lötvall J, Malmhäll C, Ekström K. Krefting Research Centre, Sahlgrenska Academy, University of Gothenburg, Sweden Mol Immunol. 2012 50(4): 278-86. Exosomes are small RNA
containing vesicles of endocytic origin, which can take part in
cell-to-cell communication partly by the transfer of exosomal RNA
between cells. Exosomes are released by many cells and can also be
found in several biological fluids including blood plasma and breast
milk. Exosomes differ compared to their donor cells not only in size
but also in RNA, protein and lipid composition. The aim of the current
study was to determine the optimal RNA extraction method for analysis
of exosomal RNA, to support future studies determining the biological
roles of the exosomal RNA. Different methods were used to extract
exosomal and cellular RNA. All methods evaluated extracted high quality
and purity RNA as determined by RNA integrity number (RIN) and OD
values for cellular RNA using capillary electrophoresis and
spectrophotometer. Interestingly, the exosomal RNA yield differed
substantially between the different RNA isolation methods. There was
also a difference in the exosomal RNA patterns in the
electropherograms, indicating that the tested methods extract exosomal
RNA with different size distribution. A pure column based approach
resulted in the highest RNA yield and the broadest RNA size
distribution, whereas phenol and combined phenol and column based
approaches lost primarily large RNAs. Moreover, the use of phenol and
combined techniques resulted in reduced yield of exosomal RNA, with a
more narrow size distribution pattern resulting in an enrichment of
small RNA including microRNA. In conclusion, the current study presents
a unique comparison of seven different methods for extraction of
exosomal RNA. As the different isolation methods give extensive
variation in exosomal RNA yield and patterns, it is crucial to select
an isolation approach depending on the research question at hand.
Biobanking of Fresh-frozen Human Colon Tissues: Impact of Tissue Ex-vivo Ischemia Times and Storage Periods on RNA Quality Bao WG, Zhang X, Zhang JG, Zhou WJ, Bi TN, Wang JC, Yan WH, Lin A. Human Tissue Bank, Taizhou Hospital of Zhejiang Province, Wenzhou Medical College, Zhejiang, China. Ann Surg Oncol. 2012 Jun 19 BACKGROUND: Biobanking
plays an important role in translational cancer research. The impact of
tissue ex-vivo ischemia time and storage period on RNA integrity is not
well documented.
METHODS: Fresh-frozen
colon tissues were collected in Taizhou Hospital of Zhejiang Province
in China since 2004. Fifty-one colon cancer tissues with tumor cell
content higher than 70 % and matched normal tissues during four storage
periods (less than 15 months, 16-20 months, 21-25 months, and 26-40
months) were chosen to detect RNA quality. Fresh colon cancer tissues
from 5 patients were cut into pieces and kept at room temperature or on
ice for 0.5, 1, 2, and 4 h before snap freezing. RNA integrity was
determined by microcapillary electrophoresis by the RNA integrity
number (RIN) algorithm.
RESULTS: Sixty-seven
percent of normal colon tissues and 94 % of colon cancer specimens
yielded RNA with a RIN of ≥7. Matched colon cancer and normal tissues
showed significant difference in RNA quality. RNA remained stable in
colon cancer tissues kept at room temperature and on ice for up to 4 h,
and long-term storage of banked colon specimens did not negatively
influence RNA quality (RNA with RIN of ≥7 banked less than 15 months,
83 %; 16-20 months, 78 %; 21-25 months, 77 %; 26-40 months, 90 %).
CONCLUSIONS: Frozen
colon tissues yield high-quality RNA in approximately 80 % of
specimens. Ex-vivo ischemia times and storage periods did not adversely
affect RNA quality. This study showed that standard operation protocols
and the maintenance of high-quality tissue repositories were the keys
to translational medicine research.
Evaluation of a novel approach for the measurement of RNA quality. Wilkes TM, Devonshire AS, Ellison SL, Foy CA. LGC, Queens Road, Teddington, Middlesex, TW11 0LY, UK BMC Res Notes. 2010 3:89. BACKGROUND: Microarray
data interpretation can be affected by sample RNA integrity. The
ScreenTape Degradation Value (SDV) is a novel RNA integrity metric
specific to the ScreenTape(R) platform (Lab901). To characterise the
performance of the ScreenTape(R) platform for RNA analysis and
determine the robustness of the SDV metric, a panel of intentionally
degraded RNA samples was prepared. These samples were used to evaluate
the ScreenTape(R) platform against an alternative approach for
measuring RNA integrity (Agilent Bioanalyzer RIN value). The samples
were also subjected to microarray analysis and the resulting data
correlated to the RNA integrity metrics.
FINDINGS: Measurement of
SDV for a panel of intentionally degraded RNA samples ranged from 0 for
intact RNA to 37 for degraded RNA, with corresponding RIN values
ranging from 10 to 4 for the same set of samples. SDV and RIN scales
both demonstrated comparable discrimination between differently treated
samples (RIN 10 to 7, SDV 0 to 15), with the SDV exhibiting better
discrimination at higher degradation levels. Increasing SDV values
correlated with a decrease in microarray sample labelling efficiency
and an increase in numbers of differentially expressed genes.
CONCLUSIONS: The
ScreenTape(R) platform is comparable to the Bioanalyzer platform in
terms of reproducibility and discrimination between different levels of
RNA degradation. The robust nature of the SDV metric qualifies it as an
alternative metric for RNA sample quality control, and a useful
predictor of downstream microarray performance.
Combined expression of miR-122a, miR-1, and miR-200b can differentiate degraded RNA samples from liver, pancreas, and stomach. Kim J, Choi NE, Oh SJ, Park SJ, Kim HK. National Cancer Center, Goyang, Republic of Korea. Pathol Int. 2011 Feb;61(2): 67-72 The effect of RNA degradation on the diagnostic utility of microRNA has not been systematically evaluated in clinical samples. We asked if the microRNA profile is preserved in degraded RNA samples derived from mouse and human tissue. We selected tissue-specific microRNA candidates from published human microarray data, and validated them using quantitative reverse transcription polymerase chain reaction (QRTPCR) analyses on flash-frozen, normal mouse liver, pancreas, and stomach tissue samples. MiR-122a, miR-1, and miR-200b were identified as tissue-specific, and the 3-microRNA-based QRTPCR could predict the tissue origin for mouse tissue samples that were left at room temperature for 2 h with an accuracy of 91.7%. When we applied this 3-microRNA predictor to clinical specimens with various degree of RNA degradation, the predictor differentiated degraded RNA samples from liver, pancreas, and stomach with an accuracy of 90% (26/29). Expression levels of miR-122a, miR-1, and miR-200b were modestly changed after the extended (2-4 h) storage at room temperature, but the magnitudes of expression changes were small compared to the expression differences between various tissues of origin. This proof-of-principle study demonstrates that RNA degradation due to extended storage at room temperature does not affect the predictive power of tissue-specific microRNA QRTPCR predictor. Evaluation of external RNA controls for the standardisation of gene expression biomarker measurements. Devonshire AS, Elaswarapu R, Foy CA. LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK. BMC Genomics. 2010 11:662. BACKGROUND: Gene expression profiling is an important approach for detecting diagnostic and prognostic biomarkers, and predicting drug safety. The development of a wide range of technologies and platforms for measuring mRNA expression makes the evaluation and standardization of transcriptomic data problematic due to differences in protocols, data processing and analysis methods. Thus, universal RNA standards, such as those developed by the External RNA Controls Consortium (ERCC), are proposed to aid validation of research findings from diverse platforms such as microarrays and RT-qPCR, and play a role in quality control (QC) processes as transcriptomic profiling becomes more commonplace in the clinical setting. RESULTS: Panels of ERCC RNA standards were constructed in order to test the utility of these reference materials (RMs) for performance characterization of two selected gene expression platforms, and for discrimination of biomarker profiles between groups. The linear range, limits of detection and reproducibility of microarray and RT-qPCR measurements were evaluated using panels of RNA standards. Transcripts of low abundance (≤ 10 copies/ng total RNA) showed more than double the technical variability compared to higher copy number transcripts on both platforms. Microarray profiling of two simulated 'normal' and 'disease' panels, each consisting of eight different RNA standards, yielded robust discrimination between the panels and between standards with varying fold change ratios, showing no systematic effects due to different labelling and hybridization runs. Also, comparison of microarray and RT-qPCR data for fold changes showed agreement for the two platforms. CONCLUSIONS: ERCC RNA standards provide a generic means of evaluating different aspects of platform performance, and can provide information on the technical variation associated with quantification of biomarkers expressed at different levels of physiological abundance. Distinct panels of standards serve as an ideal quality control tool kit for determining the accuracy of fold change cut-off threshold and the impact of experimentally-derived noise on the discrimination of normal and disease profiles. Effects of tissue handling on RNA integrity and microarray measurements from resected breast cancers. Hatzis C, Sun H, Yao H, Hubbard RE, Meric-Bernstam F, Babiera GV, Wu Y, Pusztai L, Symmans WF. Nuvera Biosciences, Woburn, MA, USA. J Natl Cancer Inst. 2011 Dec 21;103(24):1871-83. Epub 2011 Oct 27. BACKGROUND: Reliable
stability and yield of RNA from breast cancer tissues are important for
biobanking, clinical trials, and diagnostic testing.
METHODS: Aliquots of
fresh primary tumor tissue from 17 surgically resected invasive breast
cancers were placed into RNAlater at room temperature after tumor
removal (baseline) and up to 3 hours thereafter or were snap frozen at
baseline and 40 minutes thereafter. Samples were stored at -80°C
until gene expression profiling with Affymetrix Human Gene U133A
microarrays. We evaluated the effects of cold ischemic time (the time
from tumor specimen removal to sample preservation) and sample
preservation method on RNA yield, Bioanalyzer-based RNA integrity
number, microarray-based 3'/5' expression ratios for assessing
transcript integrity, and microarray-based measurement of single-gene
and multigene expression signatures. The statistical significance of
the effects was assessed using linear mixed effects regression models.
All statistical tests were two-sided.
RESULTS: Sample
preservation in RNAlater statistically significantly improved RNA
integrity compared with snap freezing as assessed by the RNA integrity
number, which increased from 7.31 to 8.13 units (difference = 0.82
units, 95% confidence interval [CI] = 0.53 to 1.11 units, P < .001),
and RNA yield, which increased threefold from 8.9 to 28.6 μg
(difference = 19.7 μg, 95% CI = 14.1 to 25.3 μg, P < .001).
Prolonged cold ischemic delay at room temperature before sample
stabilization decreased the RNA integrity number by 0.12 units/h (95%
CI = 0.02 to 0.23 units/h) compared with a projected average RNA
integrity number of 7.39 if no delays were present (P = .008) and
decreased the RNA yield by 1.5 μg/h (95% CI = 0 to 4 μg/h) from a
baseline mean RNA yield of 33.5 μg if no delays were present (P =
.019). Prolonged cold ischemia statistically significantly increased
the 3'/5' ratio of control gene transcripts, particularly of STAT1 (P
< .001). Snap freezing statistically significantly increased the
3'/5' ratio of three control transcripts (ACTB, GAPDH, and 18S rRNA).
Expression levels of single genes and multigene signatures for breast
cancer were largely unaffected by sample preservation method or cold
ischemia.
CONCLUSIONS: Sample
preservation in RNAlater improves RNA yield and quality, whereas cold
ischemia increases RNA fragmentation as measured by the 3'/5'
expression ratio of control genes. However, expression levels of single
genes and multigene signatures that are of diagnostic relevance in
breast cancer were mostly unaffected by sample preservation method or
prolonged cold ischemic duration.
Implementation of a proficiency testing for the assessment of the preanalytical phase of blood samples used for RNA based analysis. Günther K, Malentacchi F, Verderio P, Pizzamiglio S, Ciniselli CM, Tichopad A, Kubista M, Wyrich R, Pazzagli M, Gelmini S. QIAGEN GmbH, Hilden, Germany. Clin Chim Acta. 2012 Apr 11;413(7-8): 779-86 BACKGROUND: Although important improvements of downstream molecular in vitro diagnostics assays based on RNA from blood were made, the pre-analytical workflow is still poorly defined. METHODS: We performed a multicenter study within the EU-granted SPIDIA project to investigate blood collection and shipping influence on the following RNA quality parameters: yield, purity, integrity, RT-qPCR interference and IL1B, IL8, FOS and GAPDH gene expression. Two models were designed: Exp A. Ten laboratories collected blood from an own donor into two different tubes (with or without stabilizer) and extracted RNA at two different times; Exp B. Blood was drawn from a single donor and shipped to ten laboratories in two different tubes (with or without stabilizer) for RNA extraction. RESULTS: In both models and collection tubes, reliable results were obtained for purity, yield, GAPDH expression, and interferences. A substantial variation in RIN (Exp A) and in transcription levels of IL1B, IL8 and FOS (Exp B) was observed for blood collected in tube without stabilizer tubes. Overall the variability was higher among data obtained from unstabilized blood samples. CONCLUSIONS: We defined the experimental setup for a larger ring trial throughout Europe. The chosen downstream analyses verified their potential, serving as adequate markers to test the quality of blood RNA. A quantitative assay for measuring mRNA decapping by splinted ligation reverse transcription polymerase chain reaction: qSL-RT-PCR. Blewett N, Coller J, Goldstrohm A. Cellular and Molecular Biology Training Program, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. RNA. 2011 Mar;17(3): 535-543 The degradation of messenger RNA is a critical node of gene regulation. A major pathway of mRNA decay is initiated by shortening of the poly(A) tail, followed by removal of the 5' cap structure (decapping) and subsequent degradation. Decapping is an important determinate in the destruction of many transcripts. Detailed kinetic analysis of in vivo decapping rates is necessary to understand how this step is regulated. Importantly, the product of decapping is recalcitrant for investigation, in part due to its transient nature. As such, little in vivo kinetic information is available. Here we report the development of an assay that measures decapping of mRNAs by combining splinted ligation and quantitative RT-PCR (qSL-RT-PCR). We apply this method to determine the decapping rate constant for a natural mRNA in vivo for the first time. The qSL-RT-PCR assay may be adapted for use on any mRNA, providing a new tool to study regulation of mRNA decay. How the RNA isolation method can affect microRNA microarray results. Podolska A, Kaczkowski B, Litman T, Fredholm M, Cirera S. Department of Basic Animal and Veterinary Sciences, Section of Genetics and Bioinformatics, University of Copenhagen, Copenhagen, Denmark. Acta Biochim Pol. 2011; 58(4): 535-540 The quality of RNA is crucial in gene expression experiments. RNA degradation interferes in the measurement of gene expression, and in this context, microRNA quantification can lead to an incorrect estimation. In the present study, two different RNA isolation methods were used to perform microRNA microarray analysis on porcine brain tissue. One method is a phenol-guanidine isothiocyanate-based procedure that permits isolation of total RNA. The second method, miRVana™ microRNA isolation, is column based and recovers the small RNA fraction alone. We found that microarray analyses give different results that depend on the RNA fraction used, in particular because some microRNAs appear very sensitive to the RNA isolation method. We conclude that precautions need to be taken when comparing microarray studies based on RNA isolated with different methods. RNA Stability in Human Liver: Comparison of Different Processing Times, Temperatures and Methods. Lee SM, Schelcher C, Gashi S, Schreiber S, Thasler RM, Jauch KW, Thasler WE. Grosshadern Hospital Tissue Bank, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich, Germany. Mol Biotechnol. 2012 Jan 24 The accuracy of information garnered by real-time quantitative polymerase chain reaction (RT-qPCR), an important technology for elucidating molecular mechanisms of disease, is dependent on tissue quality. Thus, this study aimed to determine the effects of intra-operative manipulation, extended processing times, different temperatures or storage in RNAlater on RNA quality in liver samples for tissue banking. Liver samples, flash-frozen or in RNAlater, were collected over a time course (during surgery before blood arrest up to 1 day after surgery) with samples kept either at room temperature (RT) or on ice. This study showed that at the longest time-point at RT, the RNA quality decreased significantly by 20%. However, relative gene expressions of FOS, GUSB, MYC, HIF1α and GFER were in general not significantly different when the time-points were compared. In conclusion, samples should be kept on ice during processing, and either RNAlater or snap-freezing should be utilised for storage. Further, intra-operative manipulation and extended postoperative processing time generally does not change relative gene expression levels for the 5 genes studied, making such sampling suitable for RT-qPCR analysis. Thus, if relative gene expression of a gene of interest is stable, these guidelines will lead to increased accrual of samples to the tissue bank. Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis. França A, Melo LD, Cerca N. IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal BMC Res Notes. 2011 4:572. BACKGROUND: Microbial biofilms are communities of bacteria adhered to a surface and surrounded by an extracellular polymeric matrix. Biofilms have been associated with increased antibiotic resistance and tolerance to the immune system. Staphylococcus epidermidis is the major bacterial species found in biofilm-related infections on indwelling medical devices. Obtaining high quality mRNA from biofilms is crucial to validate the transcriptional measurements associated with the switching to the biofilm mode of growth. Therefore, we selected three commercially available RNA extraction kits with distinct characteristics, including those using silica membrane or organic extraction methods, and enzymatic or mechanical cell lysis, and evaluated the RNA quality obtained from two distinct S. epidermidis bacterial biofilms. RESULTS: RNA extracted
using the different kits was evaluated for quantity, purity, integrity,
and functionally. All kits were able to extract intact and functional
total RNA from the biofilms generated from each S. epidermidis strain.
The results demonstrated that the kit based on mechanical lysis and
organic extraction (FastRNA® Pro Blue) was the only one that was
able to isolate pure and large quantities of RNA. Normalized expression
of the icaA virulence gene showed that RNA extracted with PureLink™ had
a significant lower concentration of icaA mRNA transcripts than the
other kits tested.
CONCLUSIONS: When working with complex samples, such as
biofilms, that contain a high content extracellular polysaccharide and
proteins, special care should be taken when selecting the appropriate
RNA extraction system, in order to obtain accurate, reproducible, and
biologically significant results. Among the RNA extraction kits tested,
FastRNA® Pro Blue was the best option for both S. epidermidis
biofilms used.Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Ludyga N, Grünwald B, Azimzadeh O, Englert S, Höfler H, Tapio S, Aubele M. Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany. Virchows Arch. 2012 Feb;460(2):131-40. Epub 2012 Jan 22. Tissues used for
clinical diagnostics are mostly formalin-fixed and paraffin-embedded
(FFPE) which provides many advantages. However, the quality of the
obtained nucleic acids (NA) is reduced and this turns out to be a
challenge for further molecular analyses. Although the spectrum of
analyses of NA extracted from FFPE tissue has increased, the standard
operating procedures for NA isolation from old tissue blocks still need
to be improved. Here, we compared the efficiency of different NA
extraction methods, using FFPE tissues of variable age and origin, with
respect to downstream analyses. Our study showed that the
phenol-chloroform isoamyl alcohol (PCI) and the commercial Qiagen
protocol yielded samples with highest purity. The PCI protocol
delivered the longest amplicons even from samples from the 1970s. We
developed a short (1 h) tissue lysis procedure that turned out to be
highly time- and cost-effective when DNA quality was tested using
single and multiplex PCR. Compared to a 1-day lysis-protocol, the
amplicons were only 100 bp shorter. In addition, single-copy genes used
in daily routine were successfully amplified from long-term stored FFPE
samples following 1-h tissue-lysis. The RNA integrity numbers (RIN)
determined on RNA isolated from FFPE tissues indicated degraded RNA;
however, all RINs were above the generally agreed threshold of 1.4. We
showed that, depending on the purpose of the analysis, NA retrieved
from FFPE tissues older than 40 years may be successfully used for
molecular analysis.
Systematic discovery of structural elements governing stability of mammalian messenger RNAs. Goodarzi H, Najafabadi HS, Oikonomou P, Greco TM, Fish L, Salavati R, Cristea IM, Tavazoie S. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08540, USA Department of Molecular Biology, Princeton University, Princeton, New Jersey 08540, USA [3] Department of Biochemistry and Molecular Biophysics, and Initiative in Systems Biology, Columbia University, New York, New York 10032, USA (H.G., P.O., S.T.) The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada (H.S.N.). Nature. 2012 Apr 8 Decoding post-transcriptional regulatory programs in RNA is a critical step towards the larger goal of developing predictive dynamical models of cellular behaviour. Despite recent efforts, the vast landscape of RNA regulatory elements remains largely uncharacterized. A long-standing obstacle is the contribution of local RNA secondary structure to the definition of interaction partners in a variety of regulatory contexts, including-but not limited to-transcript stability, alternative splicing and localization. There are many documented instances where the presence of a structural regulatory element dictates alternative splicing patterns (for example, human cardiac troponin T) or affects other aspects of RNA biology. Thus, a full characterization of post-transcriptional regulatory programs requires capturing information provided by both local secondary structures and the underlying sequence. Here we present a computational framework based on context-free grammars and mutual information that systematically explores the immense space of small structural elements and reveals motifs that are significantly informative of genome-wide measurements of RNA behaviour. By applying this framework to genome-wide human mRNA stability data, we reveal eight highly significant elements with substantial structural information, for the strongest of which we show a major role in global mRNA regulation. Through biochemistry, mass spectrometry and in vivo binding studies, we identified human HNRPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1, also known as HNRNPA2B1) as the key regulator that binds this element and stabilizes a large number of its target genes. We created a global post-transcriptional regulatory map based on the identity of the discovered linear and structural cis-regulatory elements, their regulatory interactions and their target pathways. This approach could also be used to reveal the structural elements that modulate other aspects of RNA behaviour. An improved method for isolation of RNA from bone. Carter LE, Kilroy G, Gimble JM, Floyd ZE. Ubiquitin Biology Laboratory, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808, USA. BMC Biotechnol. 2012 Jan 19;12:5. BACKGROUND: Bone physiology is increasingly appreciated as an important contributor to metabolic disorders such as type 2 diabetes. However, progress in understanding the role of bone in determining metabolic health is hampered by the well-described difficulty of obtaining high quality RNA from bone for gene expression analysis using the currently available approaches. RESULTS: We developed a simple approach to isolate bone RNA that combines pulverizing the bone and the phenol-guanidinium based RNA extraction in a single step while maintaining near-freezing temperatures. This single step method increases the yield of high quality RNA by eight-fold, with RNA integrity numbers ranging from 6.7 to 9.2. CONCLUSIONS: Our streamlined approach substantially increases the yield of high-quality RNA from bone tissue while facilitating safe and efficient processing of multiple samples using readily available platforms. The RNA obtained from this method is suitable for use in gene expression analysis in real-time quantitative PCR, microarray, and next generation sequencing applications. Determination
of RNA degradation by capillary electrophoresis with cyan light-emitted
diode-induced fluorescence.
Yang TH, Chang PL. Department of Chemistry, Tunghai University, Taichung 40704, Taiwan. J Chromatogr A. 2012 Mar 30 RNA integrity plays an important role in RNA studies because poor RNA quality may have a great impact on downstream methodologies. This study proposes a cost-effective, rapid, and sensitive method for determining RNA integrity based on capillary electrophoresis that utilizes a cyan light-emitted diode-induced fluorescence as a separation tool. The capillary was initially coated with 0.1% Poly(vinylpyrrolidone) (M(ave) 1,300,000Da) to reduce electroosmotic flow and avoid RNA adsorption. When the capillary was filled with 0.4% poly(ethylene) oxide (M(ave) 4,000,000) and a nucleic acid-specific fluorescent dye, SYTO 9, the baseline separation of the 18S and 28S ribosomal RNAs (rRNAs) in total RNA was accomplished within 15min. The lowest detectable concentration for the 18S and 28S rRNAs was estimated to be 50pg/μL. Some peaks longer than the 28S rRNA that migrated slowly were observed as long as the initial total RNA concentration was optimized. The temperature-induced degradation of the large RNA fragments (longer than the 28S rRNA) was faster than that of 18S rRNA and 28S rRNA. These large RNA fragments may serve as a promising marker for testing RNA integrity compared to the traditional method. Importance of RNA isolation methods for analysis of exosomal RNA: evaluation of different methods. Eldh M, Lötvall J, Malmhäll C, Ekström K. Krefting Research Centre, Sahlgrenska Academy, University of Gothenburg, Sweden Mol Immunol. 2012 50(4):278-86. Exosomes are small RNA containing vesicles of endocytic origin, which can take part in cell-to-cell communication partly by the transfer of exosomal RNA between cells. Exosomes are released by many cells and can also be found in several biological fluids including blood plasma and breast milk. Exosomes differ compared to their donor cells not only in size but also in RNA, protein and lipid composition. The aim of the current study was to determine the optimal RNA extraction method for analysis of exosomal RNA, to support future studies determining the biological roles of the exosomal RNA. Different methods were used to extract exosomal and cellular RNA. All methods evaluated extracted high quality and purity RNA as determined by RNA integrity number (RIN) and OD values for cellular RNA using capillary electrophoresis and spectrophotometer. Interestingly, the exosomal RNA yield differed substantially between the different RNA isolation methods. There was also a difference in the exosomal RNA patterns in the electropherograms, indicating that the tested methods extract exosomal RNA with different size distribution. A pure column based approach resulted in the highest RNA yield and the broadest RNA size distribution, whereas phenol and combined phenol and column based approaches lost primarily large RNAs. Moreover, the use of phenol and combined techniques resulted in reduced yield of exosomal RNA, with a more narrow size distribution pattern resulting in an enrichment of small RNA including microRNA. In conclusion, the current study presents a unique comparison of seven different methods for extraction of exosomal RNA. As the different isolation methods give extensive variation in exosomal RNA yield and patterns, it is crucial to select an isolation approach depending on the research question at hand. Comparison of different bead-beating RNA extraction strategies: an optimized method for filamentous fungi. Leite GM, Magan N, Medina Á. Applied Mycology Group, Cranfield Health, Cranfield University, Cranfield, Bedfordshire, MK43 0AL, UK. J Microbiol Methods. 2012 88(3): 413-418 Molecular studies, especially in relation to the activity of secondary metabolite gene clusters, require the ability to extract good quality RNA from fungal biomass. This is often hindered by the cell wall structure and endogenous RNase activity in filamentous fungi. There is thus a need for rapid methods for the extraction of good quality RNA for use in microarrays and for quantitative PCR assays. The objective of this study was to examine the use of different systems for the high throughput method to extract intact RNA from filamentous fungi. Two bead beating systems with different motion patterns and speed capacities were tested in the development of the extraction protocol. They were evaluated based on the total RNA yield and overall RNA quality. The high speed bead beating with glass beads associated with an automated purification method gave more than three times higher total RNA yields with less than a quarter of the amount of mycelium required. Furthermore the integrity and overall quality was conserved, with RNA Quality Indicator (RQI) numbers consistently >7.5. This method also reduced cross contamination risks and kept RNA handling to a minimum while still being capable of multiple sample processing, reducing the time required to obtain RNA from filamentous fungi. Nucleic acid quality preservation by an alcohol-based fixative: comparison with frozen tumors in a routine pathology setting. Hostein I, Stock N, Soubeyran I, Marty M, De Mascarel I, Bui M, Geneste G, Petersen MC, Coindre JM, Macgrogan G. Department of Pathology, Institut Bergonié, Bordeaux, France Diagn Mol Pathol. 2011 20(1): 52-62 Pathologic diagnosis requires tissue fixation for histologic and immunohistologic analysis, and formalin is routinely used for this. The disadvantage of this fixative is its inability to preserve nucleic acids. Pathologic tumor diagnosis requires extensive molecular analyses, for which formalin fixation may be not adequate. Recently, an alcohol-based fixative (molecular fixative, MF) was described that allows nucleic acid preservation as well as histologic and immunohistologic studies. Moreover, the MF fixation processing system (Xpress) is fast and is well adapted to a routine process. We evaluated RNA and DNA quality within 1 month and after 1 year for 10 breast carcinomas and 20 sarcomas fixed in MF in comparison with the corresponding frozen tumors. The quality of DNA extracted from the MF-fixed tissue was similar to that extracted from the frozen tumors. The quality of RNA extracted from the MF-fixed tissue was lower than that of frozen tumors; nevertheless, a majority of RNA integrity number (RIN) values were greater than 7. Gene expression quantification by real-time polymerase chain reaction gave comparable results between tumors fixed with MF and frozen tumors. Tissue fixation at 4°C with the MF improved the RNA quality measured by the RIN value. However, after storage for 1 year at room temperature, although DNA quality was preserved, RNA extracted from tissues fixed with the MF was degraded. Tissue fixation with the MF is an important improvement for molecular pathologic diagnosis, enabling a combination of routine pathologic diagnoses and current molecular diagnoses if they are carried out near the processing time. mRNA degradation plays a significant role in the program of gene expression regulated by phosphatidylinositol 3-kinase signaling. Graham JR, Hendershott MC, Terragni J, Cooper GM. Department of Biology, Boston University, Boston, Massachusetts 02215, USA. Mol Cell Biol. 2010 Nov;30(22): 5295-305 Control of gene expression by the phosphatidylinositol (PI) 3-kinase/Akt pathway plays an important role in mammalian cell proliferation and survival, and numerous transcription factors and genes regulated by PI 3-kinase signaling have been identified. Because steady-state levels of mRNA are regulated by degradation as well as transcription, we have investigated the importance of mRNA degradation in controlling gene expression downstream of PI 3-kinase. We previously performed global expression analyses that identified a set of approximately 50 genes that were downregulated following inhibition of PI 3-kinase in proliferating T98G cells. By blocking transcription with actinomycin D, we found that almost 40% of these genes were regulated via effects of PI 3-kinase on mRNA stability. Analyses of β-globin-3' untranslated region (UTR) fusion transcripts indicated that sequences within 3' UTRs were the primary determinants of rapid mRNA decay. Small interfering RNA (siRNA) experiments further showed that knockdown of BRF1 or KSRP, both ARE binding proteins (ARE-BPs) regulated by Akt, stabilized the mRNAs of a majority of the downregulated genes but that knockdown of ARE-BPs that are not regulated by PI 3-kinase did not affect degradation of these mRNAs. These results show that PI 3-kinase regulation of mRNA stability, predominantly mediated by BRF1, plays a major role in regulating gene expression. Importance of pre-analytical steps for transcriptome and RT-qPCR analyses in the context of the phase II randomised multicentre trial REMAGUS02 of neoadjuvant chemotherapy in breast cancer patients. de Cremoux P, Valet F, Gentien D, Lehmann-Che J, Scott V, Tran-Perennou C, Barbaroux C, Servant N, Vacher S, Sigal-Zafrani B, Mathieu MC, Bertheau P, Guinebretière JM, Asselain B, Marty M, Spyratos F. Department of tumour Biology, Institut Curie, Paris 75005, France BMC Cancer. 2011 Jun 1;11:215. BACKGROUND: Identification of predictive markers of response to treatment is a major objective in breast cancer. A major problem in clinical sampling is the variability of RNA templates, requiring accurate management of tumour material and subsequent analyses for future translation in clinical practice. Our aim was to establish the feasibility and reliability of high throughput RNA analysis in a prospective trial. METHODS: This study was conducted on RNA from initial biopsies, in a prospective trial of neoadjuvant chemotherapy in 327 patients with inoperable breast cancer. Four independent centres included patients and samples. Human U133 GeneChips plus 2.0 arrays for transcriptome analysis and quantitative RT-qPCR of 45 target genes and 6 reference genes were analysed on total RNA. RESULTS: Thirty seven samples were excluded because i) they contained less than 30% malignant cells, or ii) they provided RNA Integrity Number (RIN) of poor quality. Among the 290 remaining cases, taking into account strict quality control criteria initially defined to ensure good quality of sampling, 78% and 82% samples were eligible for transcriptome and RT-qPCR analyses, respectively. For RT-qPCR, efficiency was corrected by using standard curves for each gene and each plate. It was greater than 90% for all genes. Clustering analysis highlighted relevant breast cancer phenotypes for both techniques (ER+, PR+, HER2+, triple negative). Interestingly, clustering on trancriptome data also demonstrated a "centre effect", probably due to the sampling or extraction methods used in on of the centres. Conversely, the calibration of RT-qPCR analysis led to the centre effect withdrawing, allowing multicentre analysis of gene transcripts with high accuracy. CONCLUSIONS: Our data showed that strict quality criteria for RNA integrity assessment and well calibrated and standardized RT-qPCR allows multicentre analysis of genes transcripts with high accuracy in the clinical context. More stringent criteria are needed for transcriptome analysis for clinical applications. Bleach gel: a simple agarose gel for analyzing RNA quality. Aranda PS, LaJoie DM, Jorcyk CL. Department of Biological Sciences, Boise State University, Boise, ID 83725, USA. Electrophoresis. 2012 Jan;33(2): 366-369 RNA-based applications requiring high-quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the 'bleach gel' is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality. A rapid protocol for purification of total RNA for tissues collected from pigs at a slaughterhouse. Méndez V, Avelar E, Morales A, Cervantes M, Araiza A, González D. Instituto de Ciencias Agrícolas, Universidad Autónoma de Baja California, Mexicali, México. Genet Mol Res. 2011 10(4):3251-5. Since RNA extraction is
a crucial step in many molecular techniques, the protocols for sample
collection and RNA purification need to be adapted to optimize their
performance when samples are collected from animals at commercial
facilities. Here we provide an RNA purification protocol for animal
tissues collected from slaughterhouses. This protocol, modified from
other techniques, uses TRIzol Reagent. Sample collection was performed
wearing sterile gloves and facemasks, using sterile surgical
instruments, and no longer than 8 min spent for each sample. A 0.9%
sterile sodium chloride solution was used to wash the tissue before
each sample collection. The whole process of RNA extraction was
performed under cold environment and sterile conditions. This protocol
produced good RNA yields (50 μg RNA per 100 mg tissue), good integrity
and purity (Abs(260/280) from 1.8 to 2.0), from tissues such as liver,
muscle, hypophysis, adipose tissue, and intestinal mucosa, in less than
2 h.
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