Screen And Verificate Internal Reference For The Quantification Of Circulating Micrornas

[Objective]We detect the expression level of mi RNA in the serum of the different tumor patients and healthy controls and verify in plasma samples. The suitable reference genes can be identified and validated by the q PCR detection of circulating mi RNA, and provide reference genes for the detection of circulating mi RNA cancers biomarkers and related researches.[Methods]High-throughput microarray was adopted to determine the expression of serum mi RNAs in 20 NPC patients and 20 healthy control sera, 10 cases of GC patients and 10 healthy controls after equivalent volume mixture, the mi RNAs with relative high expression levels and stability were selected for further validation. q PCR was adopted to validate the expression levels of the selected mi RNA in above 30 cancer patients and 30 healthy controls. While we analysis the suitability of U6 and mir16 as an internal reference. Ge Norm software was used to calculate and analysis the suitability values for candidate reference genes. The circulating mi RNA with relative low expression levels and stability were excluded. We selected another serum of 50 healthy persons and 178 cases of cancer patients(68 GC, each 30 colon cancer, rectal cancer, BC, 20 NPC), using q PCR to verify the expression level of the candidate reference genes. According to the comprehensive analysis and calculation of Ge Norm software, Norm Finder software, Best Keeper software, we verify the reliable and stable reference genes by screening.The stability of screened reference genes was further validated in different freezing and thawing cycle.We examined the stability of screening reference gene expression in the plasma samples.[Results]In High-throughput microarray results, we found 6 mi RNAs(hsa-mi R-191-5p, hsa-mi R-24-3p, hsa-mi R-142-3p, hsa-mi R-186-5p, hsa-mi R-135a*-3p, hsa-mi R-19b-3p) meet the requirements in serum of healthy controls,NPC and GC.According to the microarray analysis in NPC and healthy controls populations and q PCR, we found that there is a big fluctuation about U6 expression levels, also decreased expression after repeated freezing and thawing in different populations, it confirmed the U6 is not suitable as circulating mi RNA internal reference.After preliminary q PCR validation, hsa-mi R-135a*-3p was excluded for their expression not high enough with the median Ct values more than 35. The remaining 5 mi RNAs were included for further analysis. Ge Norm software applications identified above 5 candidate reference gene stability combined with its level of expression in different samples. we screened out the circulating mi RNA internal reference gene(hsa-mi R-191-5p,hsa-mi R-24-3p, hsa-mi R-142-3p).we further validate with q PCR in a certain amount of sample for screening of possible mi RNA internal reference and circulating mi RNA internal reference reported in some literature. Then Ge Norm software, Norm Finder software, Best Keeper software was Combined to comprehensive analysis.The result showed that expression of mi R-191 and mir-24 are the most stable and better than the other candidates reported mi RNA internal reference genes.In the case of repeated freezing and thawing of serum, mi R-191 and mir-24 mi RNA expression is more stable than the other.mi R-191 and mir-24 expression are relatively stable in plasma samples, suggesting that they are equally applicable to plasma mi RNA as internal reference.[Conclusions]U6 is not suitable as an internal reference for circulating mi RNA.hsa-mi R-191-5p and has-mi R-24-3p are suitable to be used as the circulating mi RNA internal control.