mRNA transcript quantification in archival samples using multiplexed, color-coded probes.
Published by BMC Biotechnol
In this recent BMC Biotechnology publication, Reis et.al., assessed the abilities of the nCounter system to accurately quantitate mRNA transcripts derived from FFPE samples using low amounts of total RNA input. To perform this study, they compared RQ-PCR and the nCounter system on matched fresh-frozen and FFPE oral carcinoma tissue samples by measuring 20 genes in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008). The genes that were assessed were COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, and RPS18. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. The authors found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r=0.90) compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r=0.50). In addition, NanoString data showed a higher mean correlation (r=0.94) between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r=0.53). The researchers concluded that “the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.”