Establishment of real-time PCR for detecting serum microRNA-21 and its preliminary application in breast cancer / 中华检验医学杂志

ABSTRACT

Objective To establish a SYBR green Ⅰ real-time PCR method for detecting serum miR-21 and preliminarily explore its value in diagnosing breast cancer.Methods Total RNA was extracted from serum by Trizol reagent.Then miR-16 ( internal reference gene for miR-21 ) and miR-21 were reverse transcribed into corresponding cDNA by stem-loop RT primers.Their cDNA were amplified and detected by using SYBR green Ⅰ real-time PCR.The accuracy of assay was analyzed by signal to noise ratio (SNR) ; the specificity of assay was evaluated by melting curve; the precision of assay was assessed by R2 of standard curve and the stability of assay was calculated by intra-assay and inter-assay variation.Furthermore,the level of miR-21 and miR-16 were detected by this method among the serum samples of 33 breast cancer patients,18 benign breast disease patients and 49 healthy individuals.And the sensitivity and specificity in breast cancer diagnosis were evaluated according to cut-off value which was defined by relative expressions of miR-21 between breast cancer patients and healthy population.Results Through optimization of temperature and time in the annealing and extension stage during PCR,SNR was ≥99.36％ ; peak of melting curve was single; R2 of standard curve was 0.994 8 and Coefficient of Variance (CV) of intra-assay ＜ 1.5％,CV of inter-assay ＜4％.They indicated that this method was accurate,specific,precise and stable.When miR-16 was taken as internal reference,the relative expressions of serum miR-21 detected by SYBR green Ⅰ realtime PCR among the serum samples of breast cancer patients,benign breast disease patients and healthy population were 20.83 ± 18.18,20.86 ± 10.11 and 9.33 ±4.44,which had statistical significance among of them (x2 =16.92,P ＜ 0.001 ).There was statistical significance in healthy people vs breast cancer patients ( Z =- 2.58,P ≤ 0.01 ) and healthy people vs benign breast disease patients ( Z =- 4.42,P ≤0.01 ),but was not between breast cancer patients and benign breast cancer patients (Z =-0.51,P =0.608).When the value of 18.32 for the relative expressions of miR-21 was defined as cut-off value,the sensitivity and specificity of this method to diagnose breast cancer were 51.5％ (17/33)and 93.9％ (46/49),respectively.Conclusions A sensitive,specific and stable SYBR green Ⅰ real-time PCR for detecting serum miR-21 has been established.This method may have some diagnostic value for breast cancer.