Real-time quantitative (PCR) applications to quantify and the expression profiles of heat shock protein (HSP70) genes in Nile tilapia, Oreochromis niloticus (L.) and Oreochromis mossambicus (P.)

摘要

Quantitative real-time PCR (qRT-PCR) has already been used to study the expression profiles of heat shock protein (Hsp) genes in Nile tilapia,?Oreochromisniloticus?(Lineaus, 1752) and?Oreochromis mossambicu?(Peter, 1983). Young fish were exposed to heat stress for 5.5 h followed by qRT-PCR of Hsp70 mRNA, using tubulin (tub) as a reference gene and flourogenic dyes. Expression of Hsp70 mRNA peaked at 1 h of heat stress and decreased at 5.5 h. This method proved to be a very sensitive technique in quantifying Hsp70 transcripts in 6.70 ng of total DNA from the two tilapia species. A standard curve was prepared, for both tilapia fishes showed almost uniform results throughout the experiment. The threshold PCR cycle (Ct), at which RT-PCR products from DNA standards accumulated to a critical level, was determined for samples in the range of 2 × 104?to 2 × 1010molecules/μl. From conclusion of this study, it is shown that when the tilapia fish muscles are subjected to stress, with the help of abiotic factors- temperatures, whether above or below optimum condition within a stipulated period of time in a peculiar way, will lead to the production of the Hsp70 protein.