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HOT PAPER -- Impact of RNA quality on reference gene expression stability.
Claudina Angela Pérez-Novo, Cindy Claeys, Frank Speleman, Paul Van Cauwenberge, Claus Bachert, and Jo Vandesompele
BioTechniques 2005 Volume 39, Number 1: pp 52-56
Gene expression quantification methods are important tools in the understanding of the molecular events underlying human diseases and in the identification of diagnostic and therapeutic targets. Generally, the messenger RNA (mRNA) used for these analyses is derived from human biopsies obtained after surgery. As a consequence, several steps during tissue handling have to be carefully controlled in order to preserve the quality and integrity of the RNA material. It is well known that RNA is sensitive to degradation by postmortem processes and inadequate sample handling or storage (1). However, RNA integrity control is often not systematically performed prior to (PCR-based) downstream analyses. While in the past, RNA quality could often not be assessed due to the limited availability of the precious sample (e.g., from microdisected cells or small biopsies), the advent of capillary gel electrophoresis and (sample retention) spectrophotometry technologies (e.g., NanoDrop® ND-1000; NanoDrop Technologies, Wilmington, DE, USA) has addressed this issue, allowing quality estimations using only nanograms (or even picograms) of total RNA (2). In addition, amplification of RNA is now an alternative method to obtain sufficient amounts to conduct gene expression studies when postmortem tissues are scarce; however, assessment of RNA quality based on the 18S and 28S ribosomal RNA bands is often not possible anymore after amplification. Furthermore, it remains to be determined whether the amplified mRNA can faithfully be used to assess RNA quality of the starting material.
Improved RNA quality and TaqMan® Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials.
Li J, Smyth P, Cahill S, Denning K, Flavin R, Aherne S, Pirotta M, Guenther SM, O'Leary JJ, Sheils O.
BMC Biotechnol. 2008 8:10.
BACKGROUND: Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62-164 bp).
RESULTS: Results from the Bioanalyzer and TaqMan data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70 degrees C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification.
CONCLUSION: Modification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts.
Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples.
Chai V, Vassilakos A, Lee Y, Wright JA, Young AH.Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis.
J Clin Lab Anal. 2005;19(5): 182-188.
One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.
Carrol ED, Salway F, Pepper SD, Saunders E, Mankhambo LA, Ollier WE, Hart CA, Day P.
BMC Immunol. 2007 8:20.
BACKGROUND: The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use.The aim of this study was to modify the PAXgene Blood RNA System kit protocol for application to small, sick children, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression.Aliquots
of 0.86 mL PAXgene reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an
in-house TaqMan assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease.
RESULTS: Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02).
CONCLUSION: We have successfully modified the PAXgene blood collection system for use in small children and demonstratedpreservation of RNA integrity and successful quantitative real-time PCR analysis.
Impact of RNA degradation on gene expression profiles: assessment of different methods to reliably determine RNA quality.
Copois V, Bibeau F, Bascoul-Mollevi C, Salvetat N, Chalbos P, Bareil C, Candeil L, Fraslon C, Conseiller E, Granci V, Mazière P, Kramar A, Ychou M, Pau B, Martineau P, Molina F, Del Rio M.
J Biotechnol. 2007 127(4): 549-559.
DNA microarray technology enables investigators to measure the expression of several 1000 mRNA species simultaneously in a biological specimen. However, the reliability of the microarray technology to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. Thus, it is of critical importance to standardize sample-handling protocols and to perform a quality assessment of RNA preparations. In this report, 59 human tissue samples were used to evaluate the relationships between RNA quality and gene expression. From Affymetrix GeneChip array data analysis of these samples, we compared the performance of the 28S/18S ratio, two computer methods (RIN and degradometer) and our in-house RNA quality scale (RQS) in assessing RNA quality. The optimal RNA reliability threshold was determined for each method using statistical discrimination measures. We showed that RQS, RIN and degradometer have a similar capacity to detect reliable RNA samples whereas the 28S/18S ratio leads to a misleading categorization. Furthermore, we developed a new approach, based on clustering analyses of full chip expression, to control RNA quality after hybridization experiments. The combination of these methods, allowing monitoring of RNA quality prior to and after the hybridization experiments, ensured reliable and reproducible microarray data.
Successful RNA extraction from various human postmortem tissues.
Heinrich M, Matt K, Lutz-Bonengel S, Schmidt U.
Int J Legal Med. 2007 121(2): 136-142.
Recently, several authors described the observation that RNA degradation does not correlate with the postmortem interval (PMI), but rather with other parameters like environmental impact and the circumstances of death. Therefore, the question arose if the analysis of gene expression could be a valuable tool in forensic genetics to contribute to the determination of the cause of death. In our study, six human tissues obtained from six individuals with PMI varying between 15 and 118 h were used for total RNA extraction. Quantification was performed using a GAPDH real-time assay, and the quality of mRNA was checked by amplification of different fragment lengths of the GAPDH transcript. In our set of samples, nearly all tissues in all PMI revealed satisfactory results, while skeletal muscle, followed by brain and heart, gave the best results. No correlation between PMI and RNA degradation could be detected, as very good results were observed for all tissues from the individual with the longest PMI. The highly promising results obtained in this study raise hopes that in the near future several fields of forensic investigation may profit from additional information about gene expression patterns and their correlation with pathological findings.
RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces.
Strand C, Enell J, Hedenfalk I, Fernö M.Preanalytical mRNA stabilization of whole bone marrow samples.
BMC Mol Biol. 2007 8: 38.
BACKGROUND: Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping.
RESULTS: Comparing the methods pair-wise, Manual vs. Ratio showed concordance (good vs. degraded RNA) in 20/24, Manual vs. RIN in 23/24, and Ratio vs. RIN in 21/24 samples. All three methods were concordant in 20/24 samples. The comparison between RNA quality and gene expression analysis showed that pieces from the same tumor and with good RNA quality clustered together in most cases, whereas those with poor quality often clustered apart. The number of samples clustering in an unexpected manner was lower for the Manual (n = 1) and RIN methods (n = 2) as compared to the Ratio method (n = 5).Assigning the data into two groups, RIN > or = 6 or RIN < 6, all but one of the top ten differentially expressed genes showed decreased expression in the latter group; i.e. when the RNA became degraded. Ontological mapping using GoMiner (p < or = 0.05; > or = 3 genes changed) revealed deoxyribonuclease activity, collagen, regulation of cell adhesion, cytosolic ribosome, and NADH dehydrogenase activity, to be the five categories most affected by RNA quality.
CONCLUSION: The results indicate that the Manual and RIN methods are superior to the Ratio method for evaluating RNA quality in fresh frozen breast cancer tissues. The objective measurement when using the RIN method is an advantage. Furthermore, the inclusion of samples with degraded RNA may profoundly affect gene expression levels.
Langebrake C, Günther K, Lauber J, Reinhardt D.
Clin Chem. 2007 53(4): 587-593.
BACKGROUND: Gene expression profiling is a useful tool for cancer diagnosis and basic research. A major limitation is that, even during short-term storage of native specimens of peripheral blood or bone marrow (BM) and/or RNA isolation, significant changes of gene expression pattern can occur because of gene induction, repression, and RNA degradation.METHODS: We investigated the
effectiveness of a newly developed RNA stabilization and preparation system for BM specimens (PAXgene Bone Marrow RNA System) over time. We analyzed 256 RNA samples, processed from 64 BM specimens.RESULTS: Although the overall RNA yield
(normalized to 1 x 10(7) leukocytes) was not different, the RNA preparation using unstabilized reference samples had an approximately 3 times higher failure rate. With the PAXgene system, we observed significantly higher RNA integrity compared with the reference RNA preparation system (P <0.01). In the stabilized samples, we found very high pairwise correlation in gene expression (DeltaDeltaC(T) 0.16-0.53) for the analyzed genes (GATA1, RUNX1, NCAM1, and SPI1) after 48-h storage compared with immediate preparation of RNA (2 h after BM collection). However, we found major differences in half of the analyzed genes using the reference RNA isolation procedure (DeltaDeltaC(T) 1.07 and 1.32).CONCLUSIONS: The PAXgene system is able to stabilize RNA from clinical BM samples and is suitable to isolate high-quality and -quantity RNA.
PAPER -- Chipping away at the chip bias: RNA degradation in microarray analysis.
POSTER -- Estimation and Reduction of the Bias Caused by RNA Degradation in Microarray Analysis.
Measurements of gene expression are based on the assumption that the analyzed RNA sample closely resembles the amount of transcripts in vivo. Established knowledge that transcripts of different genes possess different stabilities suggests that degradation of RNA occurring during the isolation procedure is also non-uniformly distributed among different RNA species. Indeed, comparison of RNA samples of different degrees of degradation shows that up to 75% of microarray-based measurements of differential gene expression can be caused by degradation bias alone1. We demonstrate that analysis of capillary-electrophoresis data does allow reproducible characterization of RNA degradation and its differentiation from apoptosis-associated RNA cleavage. Degradometer software for quantification of RNA integrity is available on our website (www.dnaarrays.org). Our results suggest that comparison of RNA samples of similar integrity eliminates skewed results of differential gene expression. Consequently, information about quantification of RNA integrity will help to improve reproducibility of microarray results.
External RNA Control Consortium (ERCC)
The External RNA Control Consortium: a progress report.
The External RNA Control Consortium (ERCC) is an ad-hoc group with approximately 70 members from private, public, and academic organizations. The group was initiated in 2003 to develop a set of external RNA control transcripts that can be used to assess technical performance in gene expression assays. The external RNA controls will be added after RNA isolation, but prior to cDNA synthesis. They are being designed to evaluate whether the results for a given experiment are consistent with defined performance criteria. All ERCC work is intended to apply to quantitative, real-time reverse transcriptase polymerase chain reaction (QRT-PCR) assays as well as one-color and two-color microarray experiments. The ERCC has worked together to define the desired properties of the transcripts, general protocols for their application, and an analysis scheme for performance assessment. In December 2003, the group developed a specification document that was discussed and refined in a public workshop at the National Institute of Standards and Technology (NIST). Protocols for the use of external RNA controls in clinical applications are included in the Molecular Methods 16-P document from the Clinical and Laboratory Standards Institute, and were developed in a formal, accredited, open, consensus forum including several ERCC members. The analysis approach was developed in a public workshop at NIST in June 2004, and is based upon the measurement of pooled transcripts at known concentrations. In the past year, the ERCC has refined specifications, generated and collected control sequences, evaluated optimal polyadenylated (polyA) tail length and identified a path forward for access and distribution of the controls, as descibed in "Proposed Methods for Testing and Selecting the ERCC External RNA Controls". Input on the proposed methods is being solicited by e-mail at firstname.lastname@example.org