Dataset Information

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue.

ABSTRACT: Whole transcriptome analysis (WTA) using RNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tissue is an invaluable tool to understand the molecular pathology of disease. RNA extracted from FFPE tissue is either degraded and/or in very low quantities hampering gene expression analysis. Earlier studies described protocols applied for cellular RNA using poly-A primer-based linear amplification. The current study describes a method, LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscriptome analysis). It employs random nonamer primer based method which can amplify short, fragmented RNA with high fidelity from as low as 5 ng to obtain enough material for WTA. The two-cycle method significantly amplified RNA at ∼1000 folds (p < 0.0001) improving the mean read lengths (p < 0.05) in WTA. Overall, increased mean read length positively correlated with on-target reads (Pearson's r = 0.71, p < 0.0001) in both amplified and unamplified RNA-seq analysis. Gene expression analysis compared between unamplified and amplified group displayed substantial overlap of the differentially expressed genes (DEGs) (log2 fold change cut-off < -2 and >2, p < 0.05) identified between lung cancer and asthma cohorts validating the method developed. This method is applicable in clinical molecular pathology field for both diagnostics and elucidation of disease mechanisms.

SUBMITTER: Bhamidimarri PM

PROVIDER: S-EPMC11234047 | biostudies-literature | 2024 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue.

Bhamidimarri Poorna Manasa PM Salameh Laila L Mahdami Amena A Abdullah Hanan Wael HW Mahboub Bassam B Hamoudi Rifat R

Heliyon 20240612 12

Whole transcriptome analysis (WTA) using RNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tissue is an invaluable tool to understand the molecular pathology of disease. RNA extracted from FFPE tissue is either degraded and/or in very low quantities hampering gene expression analysis. Earlier studies described protocols applied for cellular RNA using poly-A primer-based linear amplification. The current study describes a method, LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscript ...[more]

PMID: 38988576

Similar Datasets

Project description:BackgroundFormalin-fixed paraffin-embedded (FFPE) tissue samples are routinely archived in the course of patient care and can be linked to clinical outcomes with long-term follow-up. However, FFPE tissues have degraded RNA which poses challenges for analyzing gene expression. Next-generation sequencing (NGS) is rapidly becoming accepted as an effective tool for measuring gene expressions for research and clinical use. However, the feasibility of NGS has not been firmly established when using FFPE tissue.ResultsWe optimized strategies for whole transcriptome sequencing (RNA-seq) using FFPE tissue. Ribosomal RNA (rRNA) was successfully depleted by competitive hybridization using the Ribo-zero™ Kit (Epicentre Biotechnologies), and rRNA sequence content was less than one percent for each library. Gene expression measured by FFPE RNA-seq was compared to two different standards: RNA-seq from fresh frozen (FF) tissue and quantitative PCR (qPCR). Both FF and FFPE tumors were sequenced on an Illumina Genome Analyzer IIX with an average of 10 million reads. The distribution of FPKMs (fragments per kilobase of exon per million fragments mapped) and number of detected genes were similar between FFPE and FF. RNA-seq expressions from FF and FFPE samples from the same renal cell carcinoma (RCC) correlated highly (r = 0.919 for tumor 1 and r = 0.954 for tumor 2). On hierarchical cluster analysis, samples clustered by patient identity rather than method of preservation. TaqMan qPCR of 424 RCC-related genes correlated highly with FFPE RNA-seq expressions (r = 0.775 for FFPE tumor 1, r = 0.803 for FFPE tumor 2). Expression fold changes were considered, to assess biologic relevance of gene expressions. Expression fold changes between FFPE tumors (tumor 1/tumor 2) correlated well when comparing qPCR and RNA-seq (r = 0.890). Expression fold changes between tumors from different risk groups (our high risk RCC/The Cancer Genome Atlas, TCGA, low risk RCC) also correlated well when comparing RNA-seq from FF and FFPE tumors (r = 0.887).ConclusionsFFPE RNA-seq provides reliable genes expression data, comparable to that obtained from fresh frozen tissue. It represents a useful tool for discovery and validation of biomarkers.

Project description:BackgroundArchived tissues from previously completed prospective trials represent invaluable resource for biomarker development. However, such specimens are often stored as sections on glass slides, in which RNA is severely degraded due to prolonged air exposure. We evaluated whether a proportion of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissues yield transcriptome profiles comparable to freshly cut (FC) FFPE tissues, which can be used for retrospective class prediction analysis.MethodsGenome-wide transcriptome profiles of 6 to 7-year-old AS-FFPE tissue sections (generated from 5 to 16-year-old blocks) of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina) and digital transcript counting (nCounter) assay (NanoString), and gene signature-based prediction of HCC subclasses and prognosis was compared with previously generated FC-FFPE profiles from the same tissue blocks.ResultsRNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate to poor quality FC-FFPE samples (RNA Integrity Number: up to 2.50, R-square for technical replicates: up to 0.93). Analyzable transcriptome profiles were obtained in 64 (77%) HCC and 36 (77%) cirrhosis samples. Statistically more confident predictions based on random resampling-based method (nearest template prediction) were obtained in 37 (58%) HCC and 13 (36%) cirrhosis samples. Predictions made in FC-FFPE profiles were reproduced in 36 (97%) HCC and 11 (85%) cirrhosis AS-FFPE profiles. nCounter assay was tested in 24 cirrhosis samples, which yielded confident prediction in 15 samples (63%), of which 10 samples (67%) showed concordant predictions with FC-FFPE profiles.ConclusionsAS-FFPE tissues yielded poorer quality RNA and transcriptome profiles compared to FC-FFPE tissues. Statistically more confident class prediction was feasible in 37 of 83 HCC samples and 13 of 47 cirrhosis samples. These results suggest that AS-FFPE tissues can be regarded as a resource for retrospective transcriptome-based class prediction analysis when they are the only available materials.

Project description:BACKGROUND:Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. METHODS:Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. RESULTS:Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63-0.66) and between technical replicates (median expression difference 0.13-0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31-0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91-0.96). CONCLUSIONS:The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

Dataset Information

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue.

Publications

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets