The Stability Of Reference Genes In Mammary Gland, Liver And Small Intestine Tissue Of Mouse

This research composed of three experiments which were carried out to study the stability of reference genes in mammary gland of mouse in different lactation period, in liver of mouse after immunity treatment and in small intestine of growing mouse. The expression levels of B2M, ACTB, GAPDH, SDHA, HPRT1 and ARBP genes were detected and quantified by real-time PCR respectively. Data were analysed subsequently by geNorm algorithm, and then the suitable reference genes were chosen and applied usefully for the research of target gene in mouse.EXPERIMENT 1:Real-time quantitative PCR (qPCR) is one of the most widely used techniques for detection and quantification of mRNA expression in cells or tissues. The use of qPCR requires data normalization using internal standards such as housekeeping genes (HKGs) to obtain accurate results because of potential analytical errors due to variation. In this study, the expression levels of six potential reference genes (B2M, ACTB, GAPDH, SDHA, HPRT1 and ARBP) were investigated in mouse mammary gland by real-time qPCR using SYBR green during the different lactation days. Data were analyzed by ANOVA procedure of SAS (Version 6.12, SAS). The results showed that the expression of B2M exhibited a significant difference (P<0.05). The ranking of expression stability in these genes was (from the most stable to the least stable): GAPDH/HPRT1, ARBP, ACTB, SDHA, B2M by means of geNorm algorithm. This study suggested that the two genes GAPDH and HPRT1 may be recommented as references for normalization of real-time qPCR in mammary gland of mice in different lactation days. EXPERIMENT 2:Real-time quantitative PCR (RT-PCR) is the techniques of choice for detection and quantification of mRNA expression in cells or tissues. Suitable reference gene is essential for high precision of target gene by taking the RNA quality and efficiencies of reverse transcription into account. The purpose of the present study was to investigate the expression of B2M, ACTB, GAPDH, SDHA, HPRT1 and ARBP in mouse liver after immunity treatment by RT-PCR individually. Differences in expression levels were observed by geNorm analysis. ACTB and GAPDH were determined as suitable internal control genes. The resuts implied ACTB and GAPDH could be applied to compare the expression of target gene in mouse liver after immunity treatment.EXPERIMENT 3: Reference genes are widely used for normalization of the expression levels of mRNA in RT-PCR. But the expression of reference gene is influenced by physiological stage, tissues or cells, and the condition of experiment. The aims of this experiment were to investigate the expression of B2M, ACTB, GAPDH, SDHA, HPRT1 and ARBP by RT-PCR in small intestine of young mouse. By geNorm and normFinder analysis, SDHA and HPRT1 were finally determined as suitable reference genes used to normalize mRNA levels between different samples. The two chosen genes could be useful for research of target gene in small intestine of growing mouse.