Effect of duration of fixation with formalin on mRNA expression using quantitative RT-PCR

Area of Science:

• Molecular Biology
• Histopathology
• Biotechnology

Background:

• Analysis of messenger RNA (mRNA) in archival tissues using reverse transcription quantitative polymerase chain reaction (RT-qPCR) holds significant potential for diagnostics and research.
• Uncertainty exists regarding the feasibility of mRNA extraction and analysis from archival tissues.
• This study investigates the possibility of mRNA extraction, RT-qPCR analysis, and protocol standardization using formalin-fixed paraffin-embedded (FFPE) blocks.

Purpose of the Study:

• To assess the impact of 24-hour and 72-hour formalin fixation on mRNA quantity and quality in paraffin-embedded tissues.
• To compare the mRNA quantity and quality with that of fresh frozen tissues.
• To compare extracted mRNA expression levels using RT-qPCR across different tissue preservation methods.

Main Methods:

• Twelve tissue samples were divided into three groups: 24-hour formalin fixation, 72-hour formalin fixation, and fresh frozen (RNAlater, -80°C).
• Messenger RNA (mRNA) was extracted using the TRIzol method after routine tissue processing and wax removal for FFPE tissues.
• Extracted mRNA was quantified (Nanodrop spectrophotometer), quality-checked (mRNA TapeStation), and analyzed via cDNA synthesis followed by RT-qPCR.

Main Results:

• Satisfactory quantities of mRNA were isolated from all three preservation groups.
• Formalin-fixed tissues exhibited significantly lower mRNA quality compared to fresh frozen tissues.
• Despite quality differences, RT-qPCR results from formalin-fixed tissues were comparable to those from fresh frozen tissues (P = 0.00002).

Conclusions:

• Messenger RNA (mRNA) can be successfully extracted from archival paraffin-embedded tissue blocks.
• The extracted mRNA from FFPE tissues is suitable for extensive studies utilizing RT-qPCR.
• This validates the use of archival FFPE tissues for molecular research and diagnostics.