The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

Abstract

BackgroundUtilization 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.MethodsMatched 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.ResultsDespite 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).ConclusionsThe 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.

Targeted RNAseq assay incorporating unique molecular identifiers for improved quantification of gene expression signatures and transcribed mutation fraction in fixed tumor samples

“…Targeted RNA sequencing (targeted RNAseq) of RNA transcripts of interest provides increased sensitivity, dynamic range, reduced cost, and increased throughput compared to standard sequencing of the whole transcriptome (wtRNAseq) [10]. Although the method is compatible with RNA derived from either FF or FFPE tissue samples [11] and shows good concordance between sample types, we had observed a linear bias with higher SET ER/PR index measured from wtRNAseq, compared to the targeted RNAseq SET4 assay [5]. In this study, we updated our methods for performing the SET4 assay, including introduction of UMIs, and demonstrate the resultant increase in technical reproducibility, robustness to preanalytical and analytical conditions, and reduction of inter-platform bias between targeted and wtRNAseq.…”

“…We used residual RNA from the same samples as previously reported [5]. Briefly, those were surgically resected tissue samples collected from 12 treatment-naïve, stage I-III invasive breast cancers (IRB protocol LAB08-0824), homogenized [12], and split for processing into fresh frozen (FF) and FFPE conditions [5]. RNA was extracted from thawed FF samples using RNeasy Mini Kit (Qiagen, Hilden, Germany) [4,12].…”

“…Although most gene expression signatures are currently based on RT-PCR amplification or direct hybridization to oligonucleotide probes [1], targeted RNA sequencing (RNAseq) is developing into an alternative technology for translational research and clinical testing [2,3]. Previously, we demonstrated that the quality of gene expression quantification by whole transcriptome or targeted RNAseq measurement did not differ significantly when using three different methods (kits) to purify RNA from formalin-fixed paraffin-embedded (FFPE) samples [4,5]. Such promising analytical performance compels development of evidence-based standard operating procedures for clinical-level implementation of RNAseq on routine FFPE samples.…”

“…It also raises the possibility of whether the introduction of unique molecular identifiers (UMIs), also known as molecular barcodes, during reverse transcription could help reduce the inherent variability in amplification of target RNA sequences from FFPE tissue samples by enabling more precise enumeration of sequencing reads according to the source mRNA molecules instead of the total read counts. Thus, we modified our previously published targeted RNAseq assay that is intended to predict sensitivity to endocrinebased treatments from breast cancer samples, in order to evaluate this potential improvement in transcript quantification [5,6].…”

Stable gene expression for normalisation and single-sample scoring

“…Real time quantitative PCR (RT-qPCR) is a low throughput assay that requires smaller amounts of biological material, whereas whole transcriptome RNA-seq is a high throughput assay that requires a larger amount of RNA. In general, targeted RNA analyses tend to require smaller amounts of RNA are therefore applicable to samples where whole transcriptome analysis may not be feasible, for instance formalin fixed paraffin embedded (FFPE) samples ( 2 ). In a clinical setting, minimising the required amount of biological material is desirable as less needs to be acquired from a patient.…”