Reference Gene Selection For Expression Analysis Of Hepatic Genes Responding To Fasting Via Transcriptomic Analysis

Gene-expression analysis is increasingly important in biological research,with realtime reverse transcription PCR(RT-PCR)becoming the method of choice for highthroughput and accurate expression profiling of selected genes.And it is being increasingly used in studies of diverse biologic processes due to its outstanding accuracy,broad dynamic range,high sensitivity,and high reproducibility.However,for an exact comparison of m RNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene.In qRT-PCR analysis,systematic verification of internal reference genes is essential to produce accurate and reliable data.Ideally,expression of a reference genes should show minimal variability between samples under different experimental conditions.However,it has been gradually realized that most reference genes,especially those traditional reference genes selected based on PCR experiments of earlier days,like betaactin(ACTB)and glyceraldehyde-3-phosphate dehydrogenase(GAPDH),are not necessarily expressed at stable levels in all tissues/cells under all conditions.Therefore,it is necessary to carry out systematic experimental verification of reference genes in gene expression analysis to select appropriate reference genes.The liver is one of the most important organs in mammals.It involves many important reactions,including nutrient metabolism,detoxification and cholesterol homeostasis.Among them,glucose metabolism is tightly regulated in the liver.When the liver is damaged,blood glucose levels are out of balance,which can lead to diseases such as fatty liver disease.Therefore,the study of metabolic pathways in the liver during feeding and starvation is very important for further understanding of fatty liver,hepatitis and other liver diseases and finding treatment options.Next,through experiments,we systematically screened out genes with stable expression in the liver under feeding and starvation conditions as internal reference genes,providing further help for exploring the metabolic pathways in the liver of mice under starvation and feeding conditions.Firstly,two groups of experimental mice were constructed: feeding group and fasting group,and nutrition synchronization was given at the beginning of model construction to prevent individual differences among mice from affecting the later experimental results.In subsequent experiments,the body weight,the liver weight and the liver body ratio of mice indicated the success of the mouse model construction.Next,transcriptome sequencing was performed on liver samples from both groups of mice for further analysis.Moreover,we analyzed the relative TPM values of the reference genes commonly used in gene expression,such as β-actin,Eif4a2,GAPDH,18 S r RNA,etc.in our transcriptome data.The commonly used reference genes such as Eif4a2,Hprt,Gabarapl2,Eef1a1 and Psmc1 were up-regulated 1.6 times,1.5 times,1.4 times and 1.2 times,respectively,in the starvation group compared with the feeding group.Tuba1 a,Ppib,β-actin,Rnf181 and Tmem147 were downregulated 3.1 times,2.5 times,2.2 times,1.7 times and 1.5 times,respectively,compared with mice in the feeding group.In conclusion,the expression of commonly used reference genes was not stable in the liver of mice in the starvation group and the feeding group,so they could not be used as reference genes for gene expression analysis.We then used refinder to further analyze our transcriptome data to identify genes that were stably expressed in the livers of the starved and fed mice.Itm2 b was the most stable,followed by Mrpl43 and Eif3 b.In order to verify the genes we screened in the previous step,we used Itm2 b as reference gene to analyze gene expression levels in key pathways in liver such as peroxisome oxidation,fatty acid synthesis and gluconeogenesis by qRT-PCR.As a result,changes in gene expression levels of peroxisome oxidation,fatty acid synthesis,and gluconeogenesis in starving mice were consistent with transcriptome data,further confirming that Itm2 b is suitable as a reference gene to analyze gene expression in the study of related metabolic mechanisms in mouse liver under starvation and feeding conditions.In conclusion,Itm2 b,Mrpl43,Eif3 b genes screened from liver transcriptome data of mice in feeding group and starvation group can be used as reference genes to analyze liver gene expression of mice before and after starvation treatment.