Establishment And Application Of Cholesterol Metabolism QPCR Array

Diabetic patients tend to have dyslipidemia, which is a risk factor for atherosclerosis. Dyslipidemia can accelerate atherosclerosis, at the same time increase vascular lesions.However, the lipid metabolism is a complex biological process that involves a series of genes, the pathogenesis of diabetic dyslipidemia is unclear. There have been three kinds of technology to study the differentially expressed genes including:cDNA microarray, RNA-seq, Real-Time Quantitative PCR. These techniques have their own disadvantages:cDNA microarray is a broad spectrum screening method. So, many of the unrelated genes will produce a lot of useless or erroneous information, interfere the analysis s of result. While the ability of RNA Seq to detect all RNAs in a sample, it is a disadvantage when total RNA is examined because such a high proportion of cellular RNA arises from ribosomal and mitochondrial sources. This limits the number of reads from other RNAs and thus the number of different transcripts that can be detected and the accuracy of their expression level. The above two methods, cost much and can only be done in professional experiment center. Real-Time Quantitative PCR can only detect one or a few genes, not large-scale detection of genes. Therefore, researchers need to be able to carry out a quantitative tool for analyzing biological pathways gene, and qPCR array technology is applied.The curing of primers in the plate, which is beneficial to large-scale studies, is the critical step in the qPCR array analysis.The gene specific primers were fixed in the PCR well by high temperature. Then the PCR reaction mix and cDNA were loaded into each well of the PCR array. Real-time PCR was performed on the7500HT PCR System (Applied Biosystems) in the same reaction procedure. As compared with untreated primers, we found that the gene expression is almost unanimously in the same sample, within a preparation, the plate with cured primes can be long-term use.To evaluate the performance of the PCR array, its’amplification efficiency, reproducibility and specificity were detected. We found that qPCR array has the following characteristics:high amplification efficiency, it can be used to compare gene expression difference through relative quantitative analysis; repeatability, the average CT value was only0.2cycles difference which can detect more than2-fold change. Thus, qPCR array can be used to study the expression of expression levels of panel genes in a specific pathway or disease related genes.The study choose a total of88genes and4housekeeping genes. At the same time, mouse genomic DNA contamination primer control and negative PCR control were all included on the array. Then,the qPCR array was used to examine the mRNA expression of cholesterol metabolism-related gene to investigate the effect of high glucose on the gene expression. We found that high glucose really can affect cholesterol metabolism-related genes expression (up-regulated cholesterol synthesis, down-regulated reverse cholesterol transport), and part of the gene in a dose and time dependent.To sum up, a stable and accurate qPCR array method was established. The technology has a fast,convenient, high-throughput and high-sensitivity characteristics, It can accurately detect a panel of gene expression. So, the qPCR array can be used to study the individual level. At the same time, it is very suitable to study multi-gene complex disease..