Single Plex Branched DNA as an Alternative Assay to QuantifyP53-Like mRNA Levels in Softshell ClamMya arenariaHemocytes

Abstract

Previous studies have shown that hemic neoplasia in softshell clams is related to the level of P53 -like mRNA in hemocytes. Traditionally, the p53-like mRNA level has been quantified using quantitative real-time RT-PCR (Q RT-PCR). However, this technique requires several steps that are sources of contamination and may result in a low accuracy of the analysis. The novel aspect of this study is that the p53-like mRNA level was quantified directly from a lysate of hemocytes without any RNA extraction or reverse transcription steps. This assay is based on branched DNA (bDNA) signal amplification technology and enables quantification of p53-like mRNA levels in as few as 2,500 hemocytes, with a coefficient of variation close to 6% (range, 2–12%). A significant correlation (R2= 0.99, P ≤0.01, n= 5) was found between the p53-like mRNA quantified directly from hemocytes without RNA extraction and from 1 µg total RNA extracted from hemocytes using the classic TRIzol protocol. To compare p53-like mRNA levels in hemocytes from diseased clams collected in North River (Prince Edward Island, Canada) and from healthy clams found in Havre aux Maisons at Magdalene Island (Quebec, Canada), the quantification of relative p53-like mRNA levels was performed using our single plex assay. Data showed a significantly (P ≤0.01, n= 5) high expression of p53-like in the hemocytes of clams collected from North River. Therefore, although Q RT-PCR remains the most widely used technique for the quantification of gene expression level, we believe that single plex using bDNA technology could represent a new generation of mRNA quantification tool, enabling a more efficient mollusc health management.