# Quantifying mRNA in Highly Degraded Fixed Tissues by Nanostring Technology: A Comparative Study

## TL;DR

This study compares the effectiveness of Nanostring technology in detecting mRNA in degraded tissues fixed with Bouin’s solution versus formalin.

## Contribution

The study demonstrates that Nanostring outperforms qPCR and ddPCR in mRNA detection from highly degraded tissues.

## Key findings

- Nanostring technology detected target mRNA more effectively than ddPCR and qPCR in both FFPE and BFPE samples.
- RNA degradation still limits accurate gene expression conclusions even with Nanostring.
- BFPE tissues showed greater RNA degradation compared to FFPE tissues.

## Abstract

Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin’s solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin’s fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.