A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

Elena A Takano,Thomas Mikeska,Stephen B Fox,Alexander Dobrovic,David J Byrne

doi:10.1186/1472-6750-10-89

Elena A Takano, Thomas Mikeska + Show 3 more

Open Access

https://doi.org/10.1186/1472-6750-10-89

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Abstract

BackgroundRNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA.ResultsWe have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR) assays. This single-tube quality control (QC) assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon.ConclusionsThe multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.

Highlights

RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is chemically modified and degraded, which compromises its use in gene expression studies
We found an inverse correlation for both parameters for protocols 1 and 2 [r(P1) = -0.75, CI (95%) = -0.83 to -0.65 (P < 0.0001, n = 90) and r(P2) = -0.80, CI (95%) = -0.86 to -0.70 (P < 0.0001, n = 90), respectively]
The multiplex endpoint RT-PCR approach used in this study has been developed and optimised to assess RNA extracted from FFPE specimens for its use in RTqPCR assays

Summary

Introduction

RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is chemically modified and degraded, which compromises its use in gene expression studies. There is increasing interest in extracting RNA from these samples as the large numbers of archival FFPE samples constitute an invaluable resource for the investigation of diagnostic, prognostic or predictive disease associated alterations in gene expression (reviewed in [1]). These often represent the only diagnostic material available. RNA extracted from FFPE specimens is extensively degraded and chemically modified, which compromises its use in PCR based applications (reviewed in [2]). The addition of the monomethylol group is in principal reversible, but a considerable amount is still present after RNA purification [4]

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