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Comparative Study
, 11, 46

mRNA Transcript Quantification in Archival Samples Using Multiplexed, Color-Coded Probes

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Comparative Study

mRNA Transcript Quantification in Archival Samples Using Multiplexed, Color-Coded Probes

Patricia P Reis et al. BMC Biotechnol.

Abstract

Background: A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples.

Results: We measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18) in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008) by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR). We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. 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. We 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).

Conclusions: Based on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, 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.

Figures

Figure 1
Figure 1
Bioanalyzer assessment of RNA integrity. Representative examples of RNA integrity results after Bioanalyzer assessment of paired fresh-frozen (upper) and FFPE (lower) samples. The fresh-frozen sample shown in the upper panel had a RIN = 8.7 and the FFPE sample shown in the lower panel had a RIN = 2.3.
Figure 2
Figure 2
Correlation of results obtained from Nanostring analysis of paired fresh-frozen and FFPE tissues. Scatter plot matrix (left panel, A) for the normalized mRNA transcript quantification values obtained by Nanostring analysis of 19 fresh-frozen vs. FFPE sample pairs (n = 38 samples). In this analysis, the pair-wise Pearson product-moment correlation coefficient was 0.90 (p < 0.0001). The right panel (B) shows a heatmap analysis for the Pearson correlation of absolute mRNA transcript abundance as determined by Nanostring, for all pair-wise combinations of samples. These results show a good-high correlation between absolute mRNA transcript quantification data in fresh-frozen vs. FFPE tissues using Nanostring analysis. Fresh-frozen and FFPE tissues are interspersed, and all technical replicates are adjacent in all cases. Gene expression patterns are highly consistent among the large majority of samples.
Figure 3
Figure 3
Correlation of results obtained from RQ-PCR analysis of paired fresh-frozen and FFPE tissues. Scatter plot matrix (left panel, A) showing normalized gene expression data obtained by RQ-PCR analysis of the 19 fresh-frozen vs. FFPE sample pairs (n = 38 samples). The pair-wise Pearson product-moment correlation coefficient was 0.50 (p < 0.0001). The right panel (B) shows a heatmap analysis for the Pearson correlation of gene expression abundance as determined by RQ-PCR, for all pair-wise combinations of samples. A low-moderate correlation is observed between mRNA transcript quantification data in fresh-frozen vs. FFPE tissues, and tissues tend to cluster according to storage method.
Figure 4
Figure 4
Correlation between data obtained from Nanostring and RQ-PCR analysis on fresh-frozen and FFPE tissues. Scatter-plot matrices examining the correlation between Nanostring and RQ-PCR data in fresh-frozen (A) and FFPE (B) samples. Scatter plot matrices show normalized quantification values. The pair-wise Pearson product-moment correlation coefficient for Nanostring vs. RQ-PCR data in fresh-frozen samples was r = 0.78 (p < 0.0001); this same analysis revealed a lower correlation coefficient in FFPE samples (r = 0.59) (p < 0.0001). A corresponding heatmap for the Pearson correlation of gene expression abundance in fresh-frozen (FF) and FFPE samples using Nanostring vs. RQ-PCR is shown to the right of each scatter plot (C and D respectively). These results show a good correlation between Nanostring and RQ-PCR in fresh-frozen samples, and a lower correlation between data obtained using these two different technologies, when using clinical, archival, FFPE tissues.

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