Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy (original) (raw)
Related papers
Journal of Histochemistry & Cytochemistry, 2006
Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharidestimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed w...
A new strategy to amplify degraded RNA from small tissue samples for microarray studies
Nucleic Acids Research, 2003
RNA ampli®cation methods have been used to facilitate making probes from small tissue samples for microarray studies. Our original ampli®cation technique relied on driving the ®rst reverse transcription with oligo(dT) with a T7 RNA polymerase promoter (T7dT) on the 5¢ end, and subsequent transcriptions with random 9mers with a T3 RNA polymerase promoter (T3N9). Thus, initially, poly(A) + RNA is ampli®ed. This creates a potential problem: ampli®cations based on oligo(dT) priming could be sensitive to RNA degradation; broken mRNA strands should give rise to shorter cDNAs than those seen when intact templates are used. This would be especially troublesome when targets other than those corresponding to the 3¢ ends of transcripts are printed on an array. To solve this problem, we elected to prime cDNA synthesis with T3N9 at the beginning of each ampli®cation cycle. Following two rounds of ampli®cation, the resulting probes were comparable to those obtained with our original protocol or the Arcturus RiboAmp kit. We show below that as many as four rounds of ampli®cation can be performed reliably. In addition, as predicted, the method works well with degraded templates.
Clinical …, 2009
BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tumor material represents a valuable resource for the analysis of RNA-based biomarkers, both in research laboratories and in routine clinical testing. A robust and automated RNA-extraction method with a high sample throughput is required. METHODS: We evaluated extraction performance for 4 silica-based RNA-extraction protocols: (a) a fully automated, bead-based RNA-isolation procedure; (b) its manual counterpart; (c) a semiautomated bead-based extraction system; and (d) a manual column-based extraction kit. RNA from 360 sections (90 sections per extraction method) of 30 FFPE tumor blocks up to 20 years of age was purified and analyzed by quantitative reverse-transcription PCR for ESR1 (estrogen receptor 1), PGR (progesterone receptor), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)], and RPL37A (ribosomal protein L37a). RESULTS: The semiautomated protocol gave the best yield. The 3 bead-based methods showed good acrossmethod correlations in both yield and relative mRNA amounts (r ϭ 0.86-0.95 and 0.98, respectively). In contrast, correlations between any of the bead-based methods and the manual column-based method were worse (r ϭ 0.77-0.95 and 0.96, respectively). The fully automated method showed the lowest variation from section to section (root mean square error, 0.32-0.35 Cq, where Cq is the quantification cycle) and required the least hands-on time (1 h). CONCLUSIONS: The fully automated RNA-purification method showed the best reproducibility in gene expression analyses of neighboring sections of tissue blocks between 3 and 20 years of age and required the least overall and hands-on times. This method appears well suited for high-throughput RNA analyses in both routine clinical testing and translational research studies with archived FFPE material.
Journal of Histochemistry and Cytochemistry, 2008
S U M M A R Y Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for molecular assays on tissue after diagnostic histopathological examination. RNA from FFPE tissue suffers from strand breakage and cross-linking. Despite excellent extraction methods, RNA quality from FFPE material remains variable. To address the RNA quality factors within FFPE tissues, we studied RNA quality, isolating individual elements of the tissue fixation and processing including length of fixation in formalin and the type of buffer incorporated in the fixative. We examined the impact of the length of the tissue processing cycle as well. The optimal fixation period of 12-24 hr in phosphatebuffered formalin resulted in better-quality RNA. Longer tissue processing times were associated with higher quality RNA. We determined that the middle region of gene suffers less damage by these processes as shown by real-time quantitative RT-PCR. These data provide key information for the development of methods of analysis of gene expression in archival FFPE tissues and contribute to the establishment of objective standards for the processing and handling of tissue in surgical pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem
Optimization of Recovery of RNA From Formalin-fixed, Paraffin-embedded Tissue
Diagnostic Molecular Pathology, 2006
Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for application of diagnostic assays on tissue after microscopic examination. Not only is there a substantial archive of tissue available, but FFPE tissue remains the best method of preparation for microscopic examination in a routine clinical environment. Molecular assays, especially reverse transcription and polymerase chain reaction and expression array-based assays, offer significant potential as diagnostic, prognostic, and predictive tools, but require high quality RNA. Herein, we have optimized a reliable RNA extraction method for FFPE tissue. It is based on deparaffinization at high temperature coupled with a 3-day lysis at 651C. The average total RNA yield is 4.5 to 5.5 pg per 1 mm 3 of archival FFPE tissue, and 260/280 ratios are between 1.80 and 1.95. The extracted RNA has a modal fragment length between 100 and 200 nt by the Bioanalyzer analysis. Although modal lengths of RNA fragments were shorter, reverse transcription and polymerase chain reaction was able to amplify amplicons in range of 300 bp. Pretreatment with RNAlater followed by formalin fixation did not result in improving the RNA quality, but did improve RNA yield. Our method improves the utility of FFPE tissue for molecular profiling studies.
Evaluation of the Branched-Chain DNA Assay for Measurement of RNA in Formalin-Fixed Tissues
The Journal of Molecular Diagnostics, 2008
We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The Quan-tiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93-100%) than for qPCR (82.4-95%). Correlations between qPCR FROZEN , the gold standard , and bDNA FFPE ranged from 0.60 to 0.94 , similar to those from qPCR FROZEN and qPCR FFPE . Additionally , the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9-to 13-year-old blocks with poor RNA quality , the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion , the QuantiGene Reagent System is considerably more reliable , reproducible , and sensitive than qPCR , providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management.
Effects of RNA degradation on gene expression analysis of human postmortem tissues
Faseb Journal, 2005
The Affymetrix GeneChip platform was used to build a gene expression database of the normal human body. Postmortem human tissues represent a valuable source of biological materials for this type of study, but their use entails some delays before harvesting such tissues. We first evaluated the RNA quality obtained from tissues obtained 3-5 h postmortem and found variations that were both tissue and donor-dependent. RNAs extracted from brain regions were of higher quality than those obtained from the gut, while the cause of death was a significant factor in donor-dependent differences. To avoid these variables, we used rat duodenum to determine the effects of RNA degradation on the analysis of gene expression. Surprisingly, even samples exhibiting significant RNA degradation yielded robust gene expression results, comparable to those obtained using intact samples at a certain signal intensity cutoff. We extended these findings to our human expression database and obtained similar results, indicating that the Affymetrix platform, which is biased to the 3′ end of transcripts for detection, can tolerate significant RNA degradation, while still yielding high quality expression data. Our resulting body index expression database is a valuable research tool. As examples of potential uses, we report novel expression sites for four potential therapeutic targets-CCL27, GPR22, GPR113 and GPR128-and as well as a set of thymus-specific genes, including three not previously associated with the thymus.
Gene Expression Levels in Small Specimens From Patients Detected Using Oligonucleotide Arrays
Molecular Biotechnology, 2005
Large-scale gene expression profiling using microarray technology is often limited by the amount of tissue or cells available. A number of RNA amplification protocols have been published to overcome this problem. However, additional amplification steps can result in both a 3' bias and poor reproducibility for low abundance transcripts. We performed microarray experiments using HG-U133A GeneChip arrays to ascertain whether less than the recommended amount of RNA can be used, thus avoiding additional amplification steps. In a titration experiment, 2-10 µg of total RNA from a single cryopreserved patient specimen was used to prepare biotinylated cRNA, and the recommended standard amount of 15 µg of each preparation was used for hybridization. Statistical analysis using box plots, correlation coefficients, MvA plots, and concordance percentages revealed almost identical levels of gene expression, independent of the amount of RNA used for target preparation. Most importantly, there was no statistically significant difference when the concordance percentages for low abundance genes were compared, demonstrating that as little as 2 µg of total RNA is sufficient to perform GeneChip analysis.
Moderate degradation does not preclude microarray analysis of small amounts of RNA
BioTechniques, 2003
Gene expression analysis by microarrays using small amounts of RNA is becoming more and more popular against the background of advances and increasing importance of small-sample acquisition methods like laser microdissection techniques. The quality of RNA preparations from such samples constitutes a frequent issue in this context. The aim of this study was to assess the impact of different extents of RNA degradation on the expression profile of the samples. We induced RNA degradation in human tumor and healthy tissue samples by endogeneous ribonucleases. Next, we amplified 20 ng total RNA degraded to different extents by two rounds of in vitro transcription and analyzed them using Affymetrix oligonucleotide microarrays. Expression differences for some genes were independently confirmed by real-time quantitative PCR. Our results suggest that gene expression profiles obtained from partially degraded RNA samples with still visible ribosomal bands exhibit a high degree of similarity com...