Comparison of the QuantiGene 2.0 Assay and Real-Time RT-PCR in the Detection of p53 Isoform mRNA Expression in Formalin-Fixed Paraffin-Embedded Tissues- A Preliminary Study.
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.
Medical Genetics, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia.|Priority Research Centre for Cancer, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Newcastle, NSW, 2308, Australia.
Brianna C
BC
Morten
Medical Genetics, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia.|Priority Research Centre for Cancer, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Newcastle, NSW, 2308, Australia.|Pathology North, John Hunter Hospital, New Lambton Heights, NSW, 2305, Australia.
Rodney J
RJ
Scott
Medical Genetics, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia.|Priority Research Centre for Cancer, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Newcastle, NSW, 2308, Australia.
Kelly A
KA
Avery-Kiejda
D001940:Breast / Q000378:metabolism / Q000473:pathology*; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D015331:Cohort Studies; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D016612:Paraffin Embedding / Q000379:methods; D010865:Pilot Projects; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D020033:Protein Isoforms; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53