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

BACKGROUND

Analysis of mRNA in archival tissues using RT-qPCR has the potential to become an important element in diagnosis and research. There is uncertainty whether mRNA extraction and analyses from archival tissues are possible or not. This study will look for the possibility of mRNA extraction, RT-qPCR analysis, and standardization of the protocol using formalin fixed paraffin-embedded blocks.

OBJECTIVES

1. To determine the effect of 24-hour and 72-hour formalin-fixed paraffin-embedded tissue on the quantity and quality of mRNA. 2. To compare the quantity and quality of mRNA in fresh frozen tissues. 3. To compare the extracted mRNA expression using RT-qPCR in the above groups.

METHODOLOGY

Twelve tissue samples were collected from patients undergoing minor surgical procedures and grossed into 3 bits. Each bit was placed in 24 hours of formalin and 72 hours of formalin, and the last bit was freezed in RNAlater at -80°C (positive controls), respectively. Routine tissue processing and sectioning was done followed by wax removal for the formalin-fixed tissues, and mRNA extraction using TRIzol method was done for all three groups. Extracted mRNA was quantified using Nanodrop spectrophotometer and its quality checked on mRNA TapeStation. cDNA synthesis was done followed by RT-qPCR analysis.

RESULTS

mRNA could be isolated with satisfactory quantity in all three groups. mRNA quality was significantly low for formalin-fixed tissues. But the RT-qPCR values of the two formalin groups were comparable to those obtained in fresh frozen tissues ( value = 0.00002).

CONCLUSION

mRNA can be extracted from archives of paraffin tissue blocks that can be utilized to carry out enormous studies using RT-qPCR.