Effects Of FOXO3B Pseudogene On SNP Typing And Gene Expression Quantitative Results Of FOXO3A

Objective:Because of the 98% sequence homology in FOXO3 B pseudogene with the exon region of homo sapiens FOXO3 A gene,in some experiment such as FOXO3 A SNP genotyping and gene expression quantitative analysis,the existence of FOXO3 B gene in DNA or RNA samples will interfere with the results.In this study,we analyzed this problem to avoid erroneous experimental results due to the contamination of FOXO3 B pseudogene.Methods:1.We isolated peripheral blood mononuclear cells(PBMC)from 14 of anticoagulation blood samples,which provided kindly by Dalian Blood Center,then extracted genomic DNA(g DNA)and total RNA.2.We focused on the SNP of rs4946936,which locate in FOXO3 A exon4 region.All of the 14 samples were genotyped with PCR-restriction fragment length polymorphism(PCR-RFLP).We picked up primers from primer design software of primer3.Except one upstream primer of rs4946936-s-F,the binding sequence of all the other primers located in the FOXO3 A and FOXO3 B homologous sequence area.Rs4946936-s-F was designed at the upstream of rs4946936 area where exist 5 of base difference continuously with FOXO3 B gene.Rs4946936-s-F and rs4946936-uns-F were chose as upstream primer,shared one downstream primer of rs4946936-R for PCR-RFLP.Then we compared the rs4946936 genotypes of 14 samples acquired by different primer pair.At last,the PCR amplification fragments performed genetic sequencing to verify the results of SNP typing.3.10 of total RNA samples worked as template respectively for reverse transcription synthesis of c DNA.Then we analyzed FOXO3 A gene expression quantitatively by real-time PCR through using a set of cross-intron designed primer pair of FOXO3A-F1 and FOXO3A-R1.Both of the upstream and downstream primer were located in the homologous sequences of FOXO3 A and FOXO3 B.All of the total RNA samples for synthesis of the c DNA were eliminated g DNA contamination by g DNA eraser or not respectively.Then we compared quantitative results of FOXO3 A gene expression by using c DNA with or without g DNA elimination to find if the interference of FOXO3 B pseudogene due to g DNA contamination existed,and this interference declined due to g DNA elimination treatment.In this study,another primer pair of FOXO3A-F2 and FOXO3A-R2,which selected from a research paper[1],were chose to validate the quantitative expression results.Both of the upstream and downstream primer were located in the exon3 of FOXO3 A,and bind with FOXO3 B gene ether.4.10 of total RNA samples were selected,1 of them was rs4946936 TT type,3 of them were TC type,6 of them were CC type.After g DNA elimination treatment,the FOXO3 A gene expression was quantitatively by real-time PCR.We compared the quantitative results of the T mutant group including TC and TT type with CC type group using SPSS statistical software,and perform independent sample t test.Results:1.The rs4946936 genotype of all 14 samples identified with PCR-RFLP by using primer pair of rs4946936-s-F and rs4946936-R were completely consistence with sequencing analysis results,which means the FOXO3 B pseudogene did not affect the RFLP typing results.On the other hand,the SNP genotype acquired by using another primer pair of rs4946936-uns-F and rs4946936-R performed of FOXO3 A and FOXO3 B heterozygous type,with the interference of FOXO3 B pseudogene contamination.2.The expression of FOXO3 a gene detected by two different primer pair showed consistently higher level in g DNA elimination untreated group than treated group.Furthermore,in g DNA elimination untreated group,FOXO3 A gene expression level detected by primer pair of FOXO3A-F2 and FOXO3A-R2 was higher than that of FOXO3A-F1 and FOXO3A-R1.In addition the positive amplifying from RNA samples without g DNA elimination treatment prompted the existence of g DNA contamination,which lead to the amplifying of target gene.The PCR product size was consistent with FOXO3 A fragment.The contamination of FOXO3 A and FOXO3 B gene in RNA samples interfered with the results of gene quantitative expression analysis.No significance difference on FOXO3 A expression level detected by two primer pairs when using g DNA elimination treated sample as template.Furthermore,positive FOXO3 A PCR amplifying was not observed in RNA samples with g DNA elimination treatment,which also confirmed that the elimination of g DNA from RNA sample somehow avoided the interference of FOXO3 B pseudogene on the accuracy of FOXO3 A quantitative results.3.Among 10 samples used for gene expression analysis,there was 1 of rs4946936 TT type,3 of TC type and 6 of CC type.There was no significant difference in the level of gene expression between T mutant group(TT and TC)with CC group,p>0.05.Conclusion:1.We should pay attention to the interference of FOXO3 B pseudogene during the genotyping of FOXO3 A SNP located at exon region.According to "maximum mismatch principle",the PCR primers should be designed at the non-homological sequence area as could as possible,which can avoid the interference of the corresponding gene fragments amplified by FOXO3 B pseudogene.2.In quantitative expression analysis of FOXO3 A,because of the presence of FOXO3 B pseudogene,even using cross intron primer pair,the interference of pseudogene on the results was not avoided.The g DNA elimination treatment of RNA sample should be necessary to decline the influence of FOXO3 B on FOXO3 A m RNA quantitative results.