Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization.

Ho-Pun-Cheung A, Bascoul-Mollevi C, Assenat E, Boissière-Michot F, Bibeau F, Cellier D, Ychou M, Lopez-Crapez E.
BMC Mol Biol. 2009 Apr 15;10:31.

BACKGROUND: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard technique for mRNA quantification, but appropriate normalization is required to obtain reliable data. Normalization to accurately quantitated RNA has been proposed as the most reliable method for in vivo biopsies. However, this approach does not correct differences in RNA integrity.
RESULTS: In this study, we evaluated the effect of RNA degradation on the quantification of the relative expression of nine genes (18S, ACTB, ATUB, B2M, GAPDH, HPRT, POLR2L, PSMB6 and RPLP0) that cover a wide expression spectrum. Our results show that RNA degradation could introduce up to 100% error in gene expression measurements when RT-qPCR data were normalized to total RNA. To achieve greater resolution of small differences in transcript levels in degraded samples, we improved this normalization method by developing a corrective algorithm that compensates for the loss of RNA integrity. This approach allowedus to achieve higher accuracy, since the average error for quantitative measurements was reduced to 8%. Finally, we applied our normalization strategy to the quantification of EGFR, HER2 and HER3 in 104 rectal cancer biopsies. Taken together, our data show that normalization of gene expression measurements by taking into account also RNA degradation allows much more reliable sample comparison.
CONCLUSION: We developed a new normalization method of RT-qPCR data that compensates for loss of RNA integrity and therefore allows accurate gene expression quantification in human biopsies.

Time course analysis of RNA stability in human placenta.

Isabelle Fajardy, Emmanuelle Moitrot, Anne Vambergue, Maryse Vandersippe-Millot, Philippe Deruelle and Jean Rousseaux
Centre de Biologie Pathologie, Pôle de Biochimie et Biologie Moléculaire, CHRU de Lille, Université Lille 2, France
Service de Diabétologie et d'Endocrinologie, CHRU de Lille, France
Service de Gynécologie Obstétrique, CHRU de Lille, France
BMC Molecular Biology 2009, 10:21

Background - Evaluation of RNA quality is essential for gene expression analysis, as the presence of degraded samples may influence the interpretation of expression levels. Particularly, qRT-PCR data can be affected by RNA integrity and stability. To explore systematically how RNA quality affects qRT-PCR assay performance, a set of human placenta RNA samples was generated by two protocols handlings of fresh tissue over a progressive time course of 4 days. Protocol A consists of a direct transfer of tissue into RNA-stabilizing solution (RNAlater™) solution. Protocol B uses a dissection of placenta villosities before bio banking. We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.
Results - A long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h. The loss of the RNA integrity was higher in the placental tissues that underwent a dissection before RNA processing by comparison with those transferred directly into RNA later™ solution. That loss is moderate, with average RIN (RNA Integration Numbers) range values of 4.5–6.05, in comparison with values of 6.44–7.22 in samples directly transferred to RNAlater™ (protocol A). Among the house keeping genes tested, the B2M is the most stable.
Conclusion - This study shows that placental samples can be stored at + 4°C up to 48 h before RNA extraction without altering RNA quality. Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater™) is a prerequisite to obtain suitable RNA integrity and stability.

Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation.

Jahn CE, Charkowski AO, Willis DK.
Department of Plant Pathology, 1630 Linden drive, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Microbiol Methods. 2008 Oct;75(2): 318-324

RNA integrity is critical for successful RNA quantitation for mammalian tissues, but the level of integrity required differs among tissues. The level of integrity required for quantitation has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from Dickeya dadantii, a bacterium refractory to RNA isolation. Bacterial lysis
with Trizol using standard protocols consistently gave low RNA yields with this organism. Higher yields due to improved bacterial cells lysis was achieved with an added hot SDS incubation step, but RNA quality was low as determined by the RNA Integrity Number (RIN). Contaminating DNA remained a problem with the hot SDS-Trizol method; RNA samples required repeated, rigorous DNase treatments to reduce DNA contamination to levels sufficient for successful real-time qRT-PCR. A hot SDS-hot phenol RNA method gave the highest RNA quality and required only two DNase treatments to remove DNA. The assessment of RNA integrity using the Agilent 2100 BioAnalyzer was critical for obtaining meaningful gene expression data. RIN values below 7.0 resulted in high variation and loss of statistical significance when gene expression was analyzed by real-time qRT-PCR. We found that RNApreparations of different quality yielded drastic differences in relative gene expression ratios and led to major errors in the quantification of transcript levels. This work provides guidelines for RNA isolation and quality assessment that will be valuable for gene expression studies in a wide range of bacteria.

A comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 10(4) concentration range.

Aranda Iv R, Dineen SM, Craig RL, Guerrieri RA, Robertson JM.
Visiting Scientist, Federal Bureau of Investigation, Quantico, VA 22135; Counterterrorism and Forensic Science Research Unit, Federal Bureau of Investigation, Quantico, VA 22135.
Anal Biochem. 2009 Jan 14.

Quantification of RNA is essential for various molecular biology studies. In this work, three quantification methods were evaluated: UV absorbance, micro-capillary electrophoresis (MCE), and fluorescence-based quantification. Viral, bacterial, and eukaryotic RNA were measured in the 500-0.05 ngmul(-1)range via a ND-1000 spectrophotometer (UV), Agilent RNA 6000 kits (MCE), and Quant-iT RiboGreen assay (fluorescence). The precision and accuracy of each method were assessed and compared with a concentration independently derived using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Cost, operator time and skill,
and required sample volumes were also considered in the evaluation. Results indicate an ideal concentration range for each quantification technique to optimize accuracy and precision. The ND-1000 spectrophotometer exhibits high precision and accurately quantifies a 1 mul sample in the 500-5 ngmul(-1)range. The Quant-iT RiboGreen assay demonstrates high precision in the 1-0.05 ngmul(-1) range, but is limited to the lower RNA concentrations and is more costly than the ND-1000 spectrophotometer. The Agilent kits exhibit less precision than the ND-1000 spectrophotometer and Quant-iT RiboGreen assays in the 500-0.05 ngmul(-1)
range. However, the Agilent kits require 1 mul of sample and can determine the integrity of the RNA, a useful feature for verifying whether the isolation process was successful.

Validation of lab-on-chip capillary electrophoresis systems for total RNA quality and quantity control.

Michael W. Pfaffl, Simone Fleige, Irmgard Riedmaier
Biotechnology & Biotechnological Equipment 2008 (22): 829-834

Purity and good RNA quality are important elements for the overall success of RNA based analysis methods like microarrays and real time qRT-PCR. There are two commercially available automated systems – the Experion (Bio-Rad Laboratories) and the 2100 Bioanalyzer (Agilent Technologies) – that provide both RNA sample quality and quantity analysis. In this study different aspects like the reproducibility and sensitivity of both systems were analyzed by determining the total RNA quality and quantity extracted from various bovine tissues. Regarding quantitation, the Experion is more sensitive than the 2100 Bioanalyzer. Both systems overstate the concentration by 19-29% compared to the photometric values. For RNA quality determination, both systems show highly comparable reproducibility. With the RNA integrity number (RIN) the 2100 Bioanalyzer offers an additional opportunity to quantify the RNA quality.

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.
Deptment of Histopathology, University of Dublin, Trinity College, Dublin,
BMC Biotechnol. 2008 Feb 6;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 method of RNA isolation from paraffin blocks to assess gene expression in breast cancer.

Jarzab M, Rózanowski P, Kowalska M, Zebracka J, Rudnicka L, Stobiecka E, Jarzab B, Stachura J, Pawlega J.
Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice.
Pol J Pathol. 2008;59(2): 85-91.

Molecular oncology increasingly needs the assessment of tumor gene expression profile (transcriptome), most commonly by determination of RNA-based molecular markers employing the technique of quantitative real-time polymerase chain
reaction (Q-PCR). However, as all are methods based on RNA, to date, the experience in Q-PCR is mostly limited to freshly collected material frozen at -80 degrees C, i.e. showing no signs of RNA degradation. The aim of the present study was to implement into practice a method of RNA isolation from formalin-fixed and paraffin-embedded (FFPE) breast carcinoma samples collected during routine surgical and histopathological procedure, to further employ it in expression analysis by Q-PCR. The RNA isolation kit RNeasy FFPE (QIAGEN) was used. It was demonstrated that in samples subjected to DNAse digestion, the mean concentration of the obtained RNA was low (46 ng/microl), while during the isolation performed using solely gDNA Eliminator columns, the authors obtained RNA with an almost fourfold higher concentration value. A comparison was made between isolation
effectiveness using varying amounts of input material. It was noted that isolation efficacy was lower when three sections were employed (the concentration value of 178 ng/microl) as compared to 5-8 sections (279 and 302 ng/microl, respectively). RNA quality assessment was also performed employing the method of capillary electrophoresis by the "lab-on-a-chip" technology of Agilent
Bioanalyzer 2100. Freshly prepared material yielded in single cases samples containing RNA18S and RNA28S populations, while in samples isolated from archival paraffin blocks, the obtained RNA showed more considerable degradation, thus, was of lesser quality. In the analysis of 20 samples from the second collected series, the majority of samples were characterized by the RNA Integrity Number (RIN) values in the range of 2-2.5, still indicative of a substantial degree of RNA degradation. The mean isolation effectiveness in the second series was 885 ng/microl. In 10 of 20 blocks isolated, we succeeded in obtaining sufficient RNA
concentration, above 500 ng/microl. It was also noted that the storage time did not affect the amount of RNA obtained from a block: while isolating RNA from freshly prepared blocks, we achieved similar concentrations as when analyzing the archival material. Conclusions: the key in preserving RNA quality in paraffin blocks is the timing of material collection and fixing. Routine paraffin blocks allow for obtaining RNA for molecular studies, yet with features of considerable degradation.

Application Note - Optimizing real-time quantitative PCR experiments with the Agilent 2100 Bioanalyzer

Agilent Technologies - Steffen Mueller

This Application Note describes the benefits of the Agilent 2100 bioanalyzer in real-time quantitative PCR (qPCR) experiments. The resultsshow that the Agilent 2100 bioanalyzer is an indispensable tool to:

Measuring microRNAs: comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods.

Ach RA, Wang H, Curry B.
Agilent Laboratories, Agilent Technologies, Santa Clara, CA 95051, USA.
BMC Biotechnol. 2008 Sep 11;8: 69.

BACKGROUND: Determining the expression levels of microRNAs (miRNAs) is of great interest to researchers in many areas of biology, given the significant roles these molecules play in cellular regulation. Two common methods for measuring miRNAs in a total RNA sample are microarrays and quantitative RT-PCR (qPCR). To understand the results of studies that use these two different techniques to measure miRNAs, it is important to understand how well the results of these two analysis methods correlate. Since both methods use total RNA as a starting material, it is also critical to understand how measurement of miRNAs might be
affected by the particular method of total RNA preparation used.
RESULTS: We measured the expression of 470 human miRNAs in nine human tissues using Agilent microarrays, and compared these results to qPCR profiles of 61 miRNAs in the same tissues. Most expressed miRNAs (53/60) correlated well (R > 0.9) between the two methods. Using spiked-in synthetic miRNAs, we further examined the two miRNAs with the lowest correlations, and found the differences cannot be attributed to differential sensitivity of the two methods. We also tested three widely-used
total RNA sample prep methods using miRNA microarrays. We found that while almost all miRNA levels correspond between the three methods, there were a few miRNAs whose levels consistently differed between the different prep techniques when measured by microarray analysis. These differences were corroborated by qPCR measurements.
CONCLUSION: The correlations between Agilent miRNA microarray results and qPCR results are generally excellent, as are the correlations between different total RNA prep methods. However, there are a few miRNAs whose levels do not correlate between the microarray and qPCR measurements, or between different sample prep methods. Researchers should therefore take care when comparing results obtained using different analysis or sample preparation methods.

Focus on RNA isolation: obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue.

Wang WX, Wilfred BR, Baldwin DA, Isett RB, Ren N, Stromberg A, Nelson PT.
Sanders-Brown Center on Aging and Department of Pathology, University of Kentucky, Lexington, Kentucky 40536-0230, USA.
Biochim Biophys Acta. 2008 Nov;1779(11): 749-757

MicroRNAs (miRNAs) are present in all known plant and animal tissues and appear to be somewhat concentrated in the mammalian nervous system. Many different miRNA expression profiling platforms have been described. However, relatively little
research has been published to establish the importance of 'upstream' variables in RNA isolation for neural miRNA expression profiling. We tested whether apparent changes in miRNA expression profiles may be associated with tissue processing, RNA isolation techniques, or different cell types in the sample. RNA isolation was performed on a single brain sample using eight different RNA isolation methods, and results were correlated using a conventional miRNA microarray and then cross-referenced to Northern blots. Differing results were seen between samples obtained using different RNA isolation techniques and between microarray and Northern blot results. Another complication of miRNA microarrays is tissue-level heterogeneity of cellular composition. To investigate this phenomenon, miRNA expression profiles were determined and compared between highly-purified primary cerebral cortical cell preparations of rat primary E15-E18 neurons versus rat primary E15-E18 astrocytes. Finally, to assess the importance of dissecting human brain gray matter from subjacent white matter in cerebral cortical studies, miRNA expression profiles were compared between gray matter and immediately contiguous white matter. The results suggest that for microarray studies, cellular composition is important, and dissecting white matter from gray matter improves the specificity of the results. Based on these data, recommendations for miRNA expression profiling in neural tissues, and considerations worthy of further study, are discussed.

Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples.

Xi Y, Nakajima G, Gavin E, Morris CG, Kudo K, Hayashi K, Ju J.
Cancer Genomics Laboratory, Mitchell Cancer Institute, Mobile, Alabama 36688, USA.
RNA. 2007 Oct;13(10): 1668-1674

microRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the translational level by mainly interacting with 3' UTRs of their target mRNAs. Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery. Currently there is a lack of systematic analysis on the stability of miRNAs and optimized conditions for expression analysis using FFPE samples. In this study, the expression of miRNAs from FFPE samples was analyzed using high-throughput locked nucleic acid-based miRNA arrays. The effect of formalin fixation on the stability of miRNAs was also
investigated using miRNA real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The stability of miRNAs of archived colorectal cancer FFPE specimens was characterized with samples dating back up to 10 yr. Our results showed that the expression profiles of miRNAs were in good correlation between 1 mug of fresh frozen and 1-5 mug of FFPE samples (correlation coefficient R (2) = 0.86-0.89). Different formalin fixation times did not change the stability of miRNAs based on real-time qRT-PCR analysis. There are no significant differences of representative miRNA expression among 40 colorectal cancer FFPE specimens. This study provides a foundation for miRNA investigation using FFPE samples in cancer and other types of diseases.

Stability of RNA isolated from post-mortem tissues of Atlantic salmon (Salmo salar L.)

Seear PJ, Sweeney GE.
Biological Sciences Division, British Antarctic Survey, High Cross, Madingley Road, Cambridge CB3 0ET, UK.
Fish Physiol Biochem. 2008 34(1): 19-24

Studies of post-mortem interval on the stability of RNA from a number of various mammals have shown RNA to be stable for between 24 and 48 h following death. As yet there have been no studies looking at RNA stability in post-mortem tissues of
poikilothermic fish. Brain, kidney, liver and muscle were collected from Atlantic salmon (Salmo salar) parr and samples of each tissue were placed into RNAlatertrade mark after 0-24 h post-mortem storage at room temperature. Electrophoretic analysis of the total RNA showed degradation of ribosomal RNA only in muscle from 8 h onwards. Probing of northern blots with beta-actin showed
that, in the brain, beta-actin mRNA was stable for 24 h post-mortem but degradation of mRNA was observed after 8 h with the kidney and liver and after 4 h with the muscle. Expression of the weakly expressed thyroid hormone receptor beta (TRbeta) was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in all tissues up to 24 h post-mortem although a reduction in PCR product was observed after 8 h with muscle and 24 h with kidney. Analysis with an Agilent 2100 Bioanalyzer showed that the RNA integrity number (RIN) of brain total RNA remained constant for 8 h post-mortem with only a small fall at 24 h post-mortem. The RINs of the remaining tissues indicated degradation at 8 h post-mortem with kidney and muscle and at 24 hours post-mortem with liver. Taken together these findings show that degradation of Atlantic salmon RNA is tissue dependent but stable for at least one hour post-mortem.

Prediction of qualitative outcome of oligonucleotide microarray hybridization by measurement of RNA integrity using the 2100 Bioanalyzer capillary electrophoresis system.

Kiewe P, Gueller S, Komor M, Stroux A, Thiel E, Hofmann WK.
Department of Hematology, Oncology, and Transfusion Medicine, Charité-University Hospital Benjamin Franklin, Hindenburgdamm 30, 12203, Berlin, Germany
Ann Hematol. 2009 May 8.

RNA quality is critical to achieve valid results in microarray experiments and to save resources. The RNA integrity number (RIN) can be measured with minimal sample consumption by microfluidics-based capillary electrophoresis. To determine whether RIN can predict the qualitative outcome of microarray hybridization, we measured RIN in total RNA samples from 484 different experiments by the 2100 Bioanalyzer system and correlated with the percentage of present calls (%pc) of downstream oligonucleotide microarrays. The correlation coefficient for RNA and %pc in all 408 samples for which the bioanalyzer algorithm was able to produce an RIN was 0.475 (p < 0.05), ranging from 0.039 to 0.673 for different tissue- and assay-type subgroups. Multivariate analysis found RIN to be the best predictor of microarray quality as assessed by %pc, outperforming the 28S to 18S ratio. For a %pc threshold of 25% and 35%, we determined optimal cut points for RIN at 7.15 and 8.05, respectively. Using the suggested cut points, RIN can support the final decision whether a certain RNA sample is appropriate for successful microarray hybridization.

Removal of contaminating DNA from commercial nucleic acid extraction kit reagents.

Mohammadi T, Reesink HW, Vandenbroucke-Grauls CM, Savelkoul PH.
Sanquin Blood Bank North West Region, Amsterdam, The Netherlands.
J Microbiol Methods. 2005 May;61(2): 285-288.

Due to contamination of DNA extraction reagents, false-positive results can occur when applying broad-range real-time PCR based on bacterial 16S rDNA. Filtration of the nucleic acid extraction kit reagents with GenElute Maxiprep binding columns was effective in removing this reagent-derived contaminating DNA while the sensitivity of the assay was maintained.