Detecting The Expression By Fluorescence Level Of Bcl-2mRNAs In Intestinal Exfoliated Cells Quantitative PCR

Objective:Detect expression of Bcl-2mRNA in colorectal carcinoma, adenoma tissue, ulcerative colitis, intestinal polyps and normal mucosa organizationinvestigate the expression of Bcl-2in colorectal cancer tissue and colorectal cancer, precancerous lesions, the expression of non-colorectal cancer patientsexpression; expression and clinicopathological parameters of colorectal cancer, andto explore the expression of Bcl-2detection aids to determine the likelihood ofcolorectal cancer diagnosis and progress through analysis of Bcl-2in intestinaltissue.Detect expression of Bcl-2mRNA in colorectal carcinoma, adenoma tissue, ulcerative colitis, intestinal polyps and normal mucosa organization investigate the expression of Bcl-2in colorectal cancer tissue and colorectal cancer, precancerous lesions, the expression of non-colorectal cancer patientsexpression; expression and clinicopathological parameters of colorectal cancer, and to explorethe expression of Bcl-2detection aids to determine the likelihood of colorectalcancer diagnosis and progress through analysis of Bcl-2in intestinal tissue.Methods: Using real time PCR instrument (by SYBR Green dye method)on colorectal carcinoma, colorectal adenoma, ulcerative colitis, intestinalpolyps, and normal mucosa off the cells Bcl-2mRNA of conduct relativequantitative detection. In order to establish based on a real-time fluorescencerelative quantitative PCR technique and application of molecular diagnosis ofcolorectal cancer located on human chromosome18q21, encoding a26kDBcl-2protein is the target gene to GAPDH on chromosome12gene (Actin) asa reference gene design and synthesis of two pairs of primers, amplified by thedye method in the same reaction tube. Relative quantitative indicators△CTvalue to distinguish between patients with normal intestinal tumors. Optimizethe reaction conditions, making the concentration of the target gene and reference genes are basically the same, take2ulRNA diluted100times, and theoptical density analysis the results260nm/280nm were between1.9-2.1. Inorder to ensure the same amount of starting RNA, DEPC water to adjust RNAconcentrations. RNA adjusted in accordance with the reverse transcriptase kitmanual synthesis of cDNA.△CT value of the experiment and between, andgood repeatability, low coefficient of variation in the concentrations of RNA asa template. The relative quantification of the2-ΔΔ CtCt method to calculate therelative expression levels of Bcl-2mRNA.Result:The results suggest that: using the established method detected35cases ofcolorectal cancer patients with intestinal exfoliated cell specimens and35casesof normal intestinal exfoliated cells this normal△CT valuesrange from6.63to7.92and colorectal cancer△CTvalue range is5.92to6.81between the twogroups, there are a small number of overlapping parts, but there are significantdifferences (P <0.001); delta CT value of the colorectal adenoma group averageof4.75, the range between3.34to5.14, andcontrol group overlap, the statisticalanalysis, there were significant differences (P=0.0006<0.01); delta CT valueof the large intestine colon polyp group average of6.28, the range between5.23to6.83, the existence of a small number of overlapping part with the controlgroupby statistical analysis there a significant difference (P=0.002<0.01);delta CT value of the ulcerative colitis group average of5.25, range4.92to5.50between non-overlapping with the control group, statistical analysis ofdifferences(P=0.001<0.05);Relative quantification using the2-ΔΔ Ctmethod, the colorectal cancer groupwas1.88times the normal group, the colon polyp group is2.147times thenormal group, colorectal adenomas is6.49times the normal group, theulcerative colitis group is4.15of the normal group times. Therefore, real-timequantitative PCR△CT value of the relative quantitative method for rapid diagnosis of colorectal cancer is feasible.Conclusion：Established SYBR Green fluorescent real-time quantitativePCR method is simple, sensitive, good stability and reproducibility. Intestinalexpression of exfoliated cells, Bcl-2gene quantification and patients sufferingfrom disease states are higher than the normal intestinal relative quantitativeexpression of exfoliated cells in Bcl-2gene. The highest relative quantitativeexpression of colorectal adenomas, ulcerative colitis, colorectal polyps,colorectal cancer showed a descending order of relationship.