三种方法提取血清miRNA效果的比较
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摘要
目的比较Trizol法、蛋白酶K消化+Trizol法及沉淀裂解法提取血清miRNA的效果。方法分别选取2例健康人、2例肺炎患者和2例肺癌患者血清,以Let-7a-5p作为目标mRNA,线虫Cel-miR-39-3p作为内参,分别采用Trizol法、蛋白酶K消化+Trizol法及沉淀裂解法提取血清miRNA,以Let-7a-5p及Cel-miR-39-3p特异性引物进行反转录PCR,以反转录PCR产物为模板运用SYBGreenⅠ法进行荧光定量PCR检测。结果 Trizol法与蛋白酶K消化+Trizol法均能有效提取到血清miRNA,而沉淀裂解法由于没有对血浆中的RNA进行富集故提取效果不佳。Trizol法提取较蛋白酶K消化+Trizol法相比,可以减少蛋白酶K的消化时间且提取效果更好,并且血清与Trizol试剂的体积比为200∶600时效果相对较好。三种方法检测Cel-miR-39-3p的Ct值基本一致。Trizol法和蛋白酶K消化+Trizol法在提取过程中加入的内参丢失较少。结论 Trizol法作为总RNA提取的手段适用于血清miRNA提取,通过调整血清与Trizol试剂的用量可使提取到的血清miRNA能够满足试验需要。
Objective To compare the serum miRNA extracting efficiency of Trizol method,protease K digestion combined with Trizol method and precipitation pyrolysis method.Methods 2healthy persons,2pneumonia patients and 2lung cancer patients were enrolled in the study and their serum samples were collected.miRNA were extracted from serum samples respectively by using Trizol method,protease K digestion combined with Trizol method and precipitation pyrolysis method.Let-7a-5p was the objective miRNA of detection,with Cel-miR-39-3p as the incorporation of spike-in reference.The specific primers of Let-7a-5p(objective miRNA)and Cel-miR-39-3p(internal reference)were used in reverse transcription PCR.The products of reverse transcription PCR were used as templates for SYBGreen Ⅰfluorescence quantitative PCR assay.Results The Trizol method and protease K digestion combined with Trizol method could effectively extract serum miRNA,but precipitation pyrolysis method was not so suitable for extraction of serum miRNA.Trizol method was better than protease K digestion combined with Trizol method,which could save the proteinase K digestion time and had better extracting effects.When the volume ratio of serum and Trizol reagent was 200∶600,the extracting effect of Trizol method was best.The Ct values of Cel-miR-39-3p extracted by three methods were basically the same.There was a little loss of internal reference in the extraction process of Trizol method and protease K digestion combined with Trizol method.Conclusion The Trizol method as the extracting method of total RNA is suitable for miRNA extraction from serum.The extracted serum miRNA can meet the detection needs by adjusting the dosage of serum and Trizol reagent.
Objective To compare the serum miRNA extracting efficiency of Trizol method,protease K digestion combined with Trizol method and precipitation pyrolysis method.Methods 2healthy persons,2pneumonia patients and 2lung cancer patients were enrolled in the study and their serum samples were collected.miRNA were extracted from serum samples respectively by using Trizol method,protease K digestion combined with Trizol method and precipitation pyrolysis method.Let-7a-5p was the objective miRNA of detection,with Cel-miR-39-3p as the incorporation of spike-in reference.The specific primers of Let-7a-5p(objective miRNA)and Cel-miR-39-3p(internal reference)were used in reverse transcription PCR.The products of reverse transcription PCR were used as templates for SYBGreen Ⅰfluorescence quantitative PCR assay.Results The Trizol method and protease K digestion combined with Trizol method could effectively extract serum miRNA,but precipitation pyrolysis method was not so suitable for extraction of serum miRNA.Trizol method was better than protease K digestion combined with Trizol method,which could save the proteinase K digestion time and had better extracting effects.When the volume ratio of serum and Trizol reagent was 200∶600,the extracting effect of Trizol method was best.The Ct values of Cel-miR-39-3p extracted by three methods were basically the same.There was a little loss of internal reference in the extraction process of Trizol method and protease K digestion combined with Trizol method.Conclusion The Trizol method as the extracting method of total RNA is suitable for miRNA extraction from serum.The extracted serum miRNA can meet the detection needs by adjusting the dosage of serum and Trizol reagent.
引文
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[2]Macfarlane LA,Murphy PR.MicroRNA:biogenesis,function and role in cancer[J].Curr Genomics,2010,11(7):537-561.
[3]Bartel DP.MicroRNAs:target recognition and regulatory functions[J].Cell,2009,136(2):215-233.
[4]Esquela-Kerscher A,Slack FJ.Oncomirs-microRNAs with a role in cancer[J].Nat Rev Cancer,2006,6(4):259-269.
[5]Cortez MA,Welsh JW,Calin GA.Circulating microRNAs as noninvasive biomarkers in breast cancer[J].Recent Results Cancer Res,2012,195:151-161.
[6]Chen X,Ba Y,Ma L,et al.Characterization of microRNAs in serum:a novel class of biomarkers for diagnosis of cancer and other diseases[J].Cell Res,2008,18(10):997-1006.
[7]Wang J,Zhang KY,Liu SM,et al.Tumor-associated circulating microRNAs as biomarkers of cancer[J].Molecules,2014,19(2):1912-1938.
[8]Shen J,Stass SA,Jiang F.MicroRNAs as potential biomarkers in human solid tumors[J].Cancer Lett,2013,329(2):125-136.
[9]Cortez MA,Bueso-Ramos C,Ferdin J,et al.MicroRNAs in body fluids--the mix of hormones and biomarkers[J].Nat Rev Clin Oncol,2011,8(8):467-477.
[10]Grasedieck S,Scholer N,Bommer M,et al.Impact of serum storage conditions on microRNA stability[J].Leukemia,2012,26(11):2414-2416.
[11]Mraz M,Malinova K,Mayer J,et al.MicroRNA isolation and stability in stored RNA samples[J].Biochem Biophys Res Commun,2009,390(1):1-4.
[12]Mitchell PS,Parkin RK,Kroh EM,et al.Circulating microRNAs as stable blood-based markers for cancer detection[J].Proc Natl Acad Sci U S A,2008,105(30):10513-10518.
[13]赵德尧,杜权,郭江峰,等.血浆microRNA提取技术优化[J].浙江理工大学学报,2010,27(4):595-599.
[14]Kroh EM,Parkin RK,Mitchell PS,et al.Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR(qRT-PCR)[J].Methods,2010,50(4):298-301.
[15]Arroyo JD,Chevillet JR,Kroh EM,et al.Argonaute2complexes carry apopulation of circulating microRNAs independent of vesicles in human plasma[J].Proc Natl Acad Sci U S A,2011,108(12):5003-5008.