在福尔马林固定石蜡包埋组织中检测p53亚型mRNA表达时QuantiGene 2.0检测法与实时逆转录PCR的比较——一项初步研究
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.
作者信息
Morten Brianna C, Scott Rodney J, Avery-Kiejda Kelly A
机构信息
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.
出版信息
PLoS One. 2016 Nov 10;11(11):e0165930. doi: 10.1371/journal.pone.0165930. eCollection 2016.
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.
p53 以多种较小的异构体形式表达，其在癌症中的功能尚未完全明确。p53 异构体在不同癌症中表现出异常表达，这表明它们在调节全长 p53（FLp53）的功能方面很重要。相对 mRNA 表达的定量通常使用实时 PCR（qPCR）进行。然而，使用这种方法检测 p53 异构体存在严重局限性，特别是对于福尔马林固定石蜡包埋（FFPE）组织。使用 FFPE 肿瘤将有利于将 p53 异构体的表达与癌症的重要临床特征相关联。一种 RNA 检测的替代方法是基于杂交的 QuantiGene 2.0 检测法，已证明该方法在检测 FFPE 组织中的 RNA 方面具有优势。在这项初步研究中，我们比较了 QuantiGene 2.0 检测法和 qPCR 在匹配的新鲜冰冻（FF）和 FFPE 乳腺肿瘤中检测 FLp53 及其异构体 Δ40p53 的情况。在 FF 和 FFPE 组织中使用 qPCR 检测到了 FLp53 mRNA 表达，但 Δ40p53 mRNA 仅在 FF 组织中可检测到。QuantiGene 2.0 检测法也得到了类似结果。在 FF 组织中，两种方法显示 FLp53 相对 mRNA 表达高度相关（R2 = 0.9927，p = 0.0031），然而 Δ40p53 并非如此（R2 = 0.4429，p = 0.3345）。当比较检测 FFPE 和 FF 样本中 FLp53 mRNA 的不同方法时，使用 QuantiGene 2.0 检测法未显示相关性（R2 = 0.0002，p = 0.9863），相反，使用 qPCR 时两种组织中的表达水平高度相关（R2 = 0.8753，p = 0.0644）。这些结果表明，QuantiGene 2.0 检测法和 qPCR 方法都不足以对 FFPE 组织中的 Δ40p53 mRNA 进行定量。因此，需要替代的 RNA 检测和定量方法来研究 FFPE 样本中 Δ40p53 的相对表达。
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