Analysis Of Reference Gene Expression For Real Time PCR Ased On Relative Quantitative And Dual Spike-in Strategy In He Silkworm Bombyx Mori

Real-time quantitative PCR has become a popular method for measuring RNA levelsin recognition of its sensitivity, wide dynamic range and the potential for high samplethroughput as well as accurate quantification. Normalization strategies, in general, usereferent gene as a control. In order to avoid the introduction of experimental error,expression of this gene should not vary in response to changing conditions. However, theexpression of reference genes has been reported to vary considerably and, withoutappropriate normalization, the expression profile of a target gene could be misinterpreted.In this study, expression level of seven commonly used reference genes (ACT,GAPDH,28SrRNA, RPL3, a-Tubulin, UBC and TBP) were detected across differentdevelopment time points and across two different treatments (20-Hydroxyecdysone andRutin). The expression stability was analyzed using geNorem and NormFinder programme.We used a dual-spike-in strategy to visualize the expression level of seven reference genesand found significant variation both between normal tissues and between experimentallytreated tissues. We use GSTs1as a target gene to validate the feasibility.1．We found that the most stable reference genes were different among the differenttissues and between the different treatments. A comparison of the rankings produced by thegeNorm and NormFinder approaches revealed some differences. In three normal tissues,ACT3, GAPDH and a-Tubulin in the mid-gut, α-tubulin, UBC and TBP in the fat body aswell as a-Tubulin, ACT3and UBC in the Malpighian tubule were identified as the moststable genes. For20E induce a-Tubulin and UBC in these three tissues were established asthe most stable genes, for rutin induce ACT3and GAPDH in the fat body, and a-Tubulinand UBC in the mid gut and Malpighian tubule were established as the most stable genes.Figure3is intended to be guidance for determination of the optimal number of referencegenes. The correct choice of reference genes depends on the cells or tissues under study and researchers should look carefully at their experimental system for stable expression ofthe chosen reference gene.2. The transcription levels per gene copy of the seven reference genes were estimatedas described in Materials and Methods. The expression levels of the reference genesdetermined in three different silkworm tissues and under differents treatments are shown asa logarithmic histogram in Figures. The expression levels of these genes were not constant;P value﹤0.01was deemed to be significant variation. For all seven genes, the transcriptionlevels were quite different at different stages of development.and under differenttreatments.3. Two normalization methods, the relative quantitation method and the dual spike-inqPCR method were used to assess the profiling of GSTs1in fat body tissue treated with20E and with rutin. The relative expression and transcription level per gene copy of GSTs1in FB were measured. The relative expression level of a target gene is attributed to variableexpression of the reference genes. The use of different reference genes led to differentexperimental results. The profiling of GSTs1varied according to the normalization methodused; the relative quantitation and dual spike-in qPCR techniques yielded different results.Using dual spike-in qPCR method seems more reasonable.4. The results of GST protease activity test showed that there is certain relevancebetween the gene transcription level and protein expression level, but is not absolutelyrelevance.The silkworm B. mori is an economically important insect and has become a modelorganism in the study of lepidopteran and arthropod biology. Because of its typical insectlife-cycle, its signal transduction, protein synthesis and metabolism change rapidly andsignificantly among different development time points, the highly tissue-specific results infunctional differences. This means that the reference genes in the silkworm cannot bestable, unlike mammalian reference genes. Usually, reference genes in the silkworm havebeen selected on the basis of consensus and experience in other organisms rather thanempirical evidence in support of their efficacy. Data were analysed subsequently bygeNorm and algorithm, and then the suitable reference genes were chosen and appliedusefully for the research of target gene in silkworm while use relative quantitative. On theother side the dual spike-in PCR technique uses exogenous reference mRNA along withmRNA absolute standard curves and appears to give reliable results. It can promote the development of real-time quantitative PCR technology innovative at a certain extent.