Reliable quantification of mRNA in archived formalin-fixed tissue with or without paraffin embedding

Abstract

Introduction: Formalin fixation and paraffin embedding (FFPE) is a standard method for tissue sample storage and preservation in pathology archives. The Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) is a useful method for gene expression analysis, but its sensitivity is significantly decreased in FFPE tissue due to the fixation process. This process results in chemical modifications of RNA, cross-links proteins to RNA, and degrades RNA in these archived samples, hindering the reverse transcription step of the conventional RT-pPCR method and preventing generation of a cDNA that is long enough for the subsequent quantitative PCR step.

Methods: In this study, we used a multi-species RT-qPCR method originally developed to detect mRNA in tissue homogenate samples (Wang et al., 2011) and applied it to effectively detect a specific mRNA in formalin-fixed tissues with or without paraffin-embedding by targeting mRNA sequences as short as 24 nucleotides.

Results: Target sizes ranging from 24 to 91 nucleotides were evaluated using this multi-species RT-qPCR assay. Data generated with FFPE tissues demonstrated that use of short target sequences relieved the dependence on RNA quality and could reliably quantify mRNA. This method was highly sensitive, reproducible, and had a dynamic range of five orders of magnitude. Importantly, this method could quantify mRNA in prolonged formalin-fixed and FFPE tissue, where conventional RT-qPCR assays failed. Moreover, a similar result for small interfering RNA (siRNA)-mediated Apob mRNA knockdown was obtained from tissues fixed in formalin solution for 3months to 4years, and was found to be comparable to results obtained with frozen liver tissues.

Discussion: Therefore, the method presented here allows for preclinical and clinical retrospective and prospective studies on mRNA derived from archived FFPE and prolonged formalin-fixed tissue.

Keywords: FFPE; Prolonged formalin-fixed tissue; RT-qPCR; mRNA quantification methods; siRNA-mediated mRNA knockdown.