Abstract
p53 is expressed as multiple smaller isoforms whose functions in cancer are not well understood. The p53 isoforms demonstrate abnormal expression in different cancers, suggesting they are important in modulating the function of full-length p53 (FLp53). The quantification of relative mRNA expression has routinely been performed using real-time PCR (qPCR). However, there are serious limitations when detecting p53 isoforms using this method, particularly for formalin-fixed paraffin-embedded (FFPE) tissues. The use of FFPE tumours would be advantageous to correlate expression of p53 isoforms with important clinical features of cancer. One alternative method of RNA detection is the hybridization-based QuantiGene 2.0 Assay, which has been shown to be advantageous for the detection of RNA from FFPE tissues. In this pilot study, we compared the QuantiGene 2.0 Assay to qPCR for the detection of FLp53 and its isoform Δ40p53 in matched fresh frozen (FF) and FFPE breast tumours. FLp53 mRNA expression was detected using qPCR in FF and FFPE tissues, but Δ40p53 mRNA was only detectable in FF tissues. Similar results were obtained for the QuantiGene 2.0 Assay. FLp53 relative mRNA expression was shown to be strongly correlated between the two methods (R2 = 0.9927, p = 0.0031) in FF tissues, however Δ40p53 was not (R2 = 0.4429, p = 0.3345). When comparing the different methods for the detection of FLp53 mRNA from FFPE and FF samples, no correlation (R2 = 0.0002, p = 0.9863) was shown using the QuantiGene 2.0 Assay, and in contrast, the level of expression was highly correlated between the two tissues using qPCR (R2 = 0.8753, p = 0.0644). These results suggest that both the QuantiGene 2.0 Assay and qPCR methods are inadequate for the quantification of Δ40p53 mRNA in FFPE tissues. Therefore, alternative methods of RNA detection and quantification are required to study the relative expression of Δ40p53 in FFPE samples.
Highlights
Formalin-fixed paraffin embedded (FFPE) tissue samples have been collected for thousands of breast cancer specimens with substantial clinical information for diagnostic investigation
Our results showed that the QuantiGene 2.0 Assay was not comparable to real-time PCR for the detection of full-length p53 (FLp53) mRNA from FFPE samples, compared to matched fresh frozen (FF) tissue, and that Δ40p53 mRNA expression could not be quantitated using either method
There were clear differences in the detected signal for Δ40p53 mRNA, where Δ40p53 was detected in the FF samples using both methods, but was unable to be detected using real-time PCR in the FFPE samples (Fig 1B, Table 2, S2 and S3 Figs)
Summary
Formalin-fixed paraffin embedded (FFPE) tissue samples have been collected for thousands of breast cancer specimens with substantial clinical information for diagnostic investigation. Advances in mRNA quantitation are providing novel opportunities for quantitative gene expression analysis in FFPE tissues One such method is the QuantiGene 2.0 Assay by Panomics [6]. The probe-based assay uses Z-probe structures which binds to smaller 20bp regions consecutively to generate a larger targeted region, compared to a primer-probe amplicon used in real-time PCR This assay is advantageous for the detection of highly degraded RNA from FFPE tissues, as the manner in which the probes consecutively bind to the sequence allows for high specificity, and it does not require any reverse transcription or mRNA processing and avoids any loss of signal, or inaccuracies, that may result from chemical modification or degraded material caused by tissue fixation [4]
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