Comparison Of MicroRNA-21in Breast Cancer Tissue Detection Results With Two Fluorescent Quantitative PCR Methods

Objective:Application SYBR Green I fluorescent dyes method and TaqMan fluorescence probe method to detect breast cancer tissue miR-21, Through the comparison of the two methods, We expect to select the higher precision, relatively lower cost, more applicable to clinical miRNA test methods.Methods:Here we Collect A total of14cases of breast cancer and adjacent tissues which was admitted into the first affiliated hospital of Zun Yi Medical college in2010as material for extracting total RNA, design miR-21specific primers and U6primer reverse transcription into the first cDNA, respectively with the general type fluorescent dye SYBR Green I and high specificity for TaqMan fluorescent probe as indicator, real-time fluorescent quantitative PCR for detection of miR-21in breast cancer and adjacent tissues in the relative levels of expression. Application of statistical analysis of SYBR Green I dye method and TaqMan probe assay for detection of the difference between the final results, using Northern Blot method to conduct the experimental verification.Results:Extraction of total RNA by1%agarose gel electrophoresis revealed marked28s and18S rRNA, and OD260/OD280ratio betweenl.8-2.0, it reveals higher quality of the RNA that we have extracted; The melting curve showed that U6and miR-21melting curves of single, shows its good specificity. The amplification curve of U6and miR-21accords with PCR amplification rule. By statistical analysis, SYBR Green I dyes method for the detection of cancer and adjacent tissues in miR-21, paired t test showed significant differences between the groups (t=6.704, P<0.001); Taqman probe method comparison between groups, the expression had significant difference (t=7.238, P<0.001);in view of two quantitative PCR methods for the detection of breast tissue in the expression of miR-21, there is no significant difference (P>0.05) through analysis of variance results. Northern Blot experimental results showed that the results were reliable. Conclusion:1.In the amplification products of specific conditions, the application of SYBR Green I dyes method for the detection of microRNA sensitivity and specificity and TaqMan probe method is similar, the results had no significant difference between. SYBR Green I dye method is a more simple、effective、practical, wider application range, relatively lower cost, more applicable to clinical detection method miRNA.2.miR-21expression increases in breast cancer,compared with paracancerous tissue, there were significant differences (P<0.05). MiR-21expression and breast cancer have a certain correlation.