血清miRNA-221-3p标准品的制备及其绝对含量测定
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摘要
目的:构建miRNA-221-3p的标准品,并检测其血清绝对含量,为今后进一步明确检测血清miRNA-221-3p在辅助判断疾病中的潜在价值和意义。方法:设计miRNA-221-3p特异性引物,提取样品血清总RNA,反转录获得含miRNA-221-3p特异性引物c DNA。通过PCR获得含有miRNA-221-3p的DNA片段,然后置入到pc DNA3.1质粒中。经测序确定重组质粒pc DNA3.1(pc DNA3.1-miRNA-221-3p)构建正确。通过PCR从pc DNA3.1-miRNA-221-3p中获得含有T7启动子和miRNA-221-3p的基因片段,然后用体外转录得到miRNA-221-3p的标准品,最后计算出标准品的浓度(copies/μg RNA)。取miRNA-221-3p标准品反转录进行梯度稀释实时荧光定量PCR,根据标准品的浓度和CT值绘制标准曲线。结果:miRNA-221-3p重组质粒构建成功,体外转录得到纯化的miRNA-221-3p标准品。标准曲线公式为:Y=-0.255X+12.543,相关系数R2=0.9999,X代表Ct值,Y代表样品的含量(copies)的lg值。结论:成功获得血清miRNA-221-3p标准品,miRNA-221-3p标准品绝对含量(copies/μg RNA)等于3.49214E+12e-0.5871CT值。
Objective: To prepare serum mi R-221-3 p standard substance, to determine its absolute content,and to investigate the value and significance of serum mi R-221-3 p determination in disease diagnosis. Methods:The specific primer of mi R-221-3 p was designed, total serum RNA was extracted, and then reverse transcription was performed to obtain c DNA that contained the specific primer of mi R-221-3 p. PCR was performed to obtain DNA fragments containing mi R-221-3 p, which were inserted into pc DNA3.1 plasmid. Sequencing was used to confirm whether the recombinant plasmid pc DNA3.1-mi R-221-3 p was correctly constructed. Then PCR was used to obtain gene fragments containing T7 promoter and mi R-221-3 p from pc DNA3.1-mi R-221-3 p, and in vitro transcription was performed to obtain mi R-221-3 p standard substance. The concentration of mi R-221-3 p standard substance(copies/μg RNA) was calculated. Gradient dilution and quantitative real-time PCR were performed for mi R-221-3 p standard substance, and a standard curve was plotted based on concentration and CT of standard substance. Results: The recombinant plasmid of mi R-221-3 p was constructed successfully, and the purified mi R-221-3 p standard substance was obtained by in vitro transcription. The standard curve formula was Y =-0.255 X + 12.543(R2= 0.9999), with X standing for threshold cycle and Y for the lg value of content.Conclusions: Serum mi R-221-3 p standard substance is obtained successfully, and the absolute content of mi R-221-3 p standard substance equals to 3.49214 E + 12 e-0.5871 CT.
Objective: To prepare serum mi R-221-3 p standard substance, to determine its absolute content,and to investigate the value and significance of serum mi R-221-3 p determination in disease diagnosis. Methods:The specific primer of mi R-221-3 p was designed, total serum RNA was extracted, and then reverse transcription was performed to obtain c DNA that contained the specific primer of mi R-221-3 p. PCR was performed to obtain DNA fragments containing mi R-221-3 p, which were inserted into pc DNA3.1 plasmid. Sequencing was used to confirm whether the recombinant plasmid pc DNA3.1-mi R-221-3 p was correctly constructed. Then PCR was used to obtain gene fragments containing T7 promoter and mi R-221-3 p from pc DNA3.1-mi R-221-3 p, and in vitro transcription was performed to obtain mi R-221-3 p standard substance. The concentration of mi R-221-3 p standard substance(copies/μg RNA) was calculated. Gradient dilution and quantitative real-time PCR were performed for mi R-221-3 p standard substance, and a standard curve was plotted based on concentration and CT of standard substance. Results: The recombinant plasmid of mi R-221-3 p was constructed successfully, and the purified mi R-221-3 p standard substance was obtained by in vitro transcription. The standard curve formula was Y =-0.255 X + 12.543(R2= 0.9999), with X standing for threshold cycle and Y for the lg value of content.Conclusions: Serum mi R-221-3 p standard substance is obtained successfully, and the absolute content of mi R-221-3 p standard substance equals to 3.49214 E + 12 e-0.5871 CT.
引文
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12.闫继红,杨姝,孙海梅,等.共沉默mi R-221-3p/222-3p表达抑制骨髓间充质干细胞增殖及促进软骨分化[J].中国组织工程研究,2015,15(50):8 056-8 061.
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15.Cheng Y,Zhang X,Li Z,et al.Highly sensitive determination of micro RNA using target-primed and branched rolling-circle amplification[J].Angew Chem Int Ed Engl,2009,48(18):3 268-3 272.
2.闫丽,张瑶楠,张蔚,等.优化和验证人精浆mi RNAs提纯方法[J].生殖医学杂志,2017,26(6):568-572.
3.Brown PN,Yin H.PNA-based micro RNA inhibitors elicit anti-inflammatory effects in microglia cells[J].Chemical Communications,2013,49(39):4 415-4 417.
4.Luna-Aguirre CM,Martinez-Fierro LM,Maraguilar F,et al.Circulating micro RNA expression profile in Bcell acute lymphoblastic leukemia[J].Cancer Biomark,2015,15(3):299-310.
5.Kan AA,Van Erp S,Derijck AA,et al.Genome-wide micro RNA profiling of human temporal lobe epilepsy identifies modulators of the immune response[J].Cellular and Molecular Life Sciences,2012,69(18):3 127-3 145.
6.李梦萍,曹海明,武哲丽,等.原发性肝癌不同血瘀证患者肝组织微小RNA表达差异的初步研究[J].山东医药,2014,54(38):1-4.
7.陈雪梅,丁雨,吴思思.两种实时定量聚合酶链反应方法检测血浆微量微RNA的比较[J].华西医学,2017,032(5):699-704.
8.刘金立,刘淑阁,熊倩,等.急性单核细胞白血病特异性mi RNA分子标志物的鉴定[J].温州医科大学学报,2016,46(10):703-708,715.
9.Slagsvold KH,Rognmo O,Hoydal MA,et al.Remote ischemic preconditioning preserves mitochondrial function and influences myocardial micro RNA expression in atrial myocardium during coronary bypass surgery[J].Circ Res,2014,114(5):851-859.
10.Peng JS,Sarkar S,Chang SL.Opioid receptor expression in human brain and peripheral tissues using absolute quantitative real-time RT-PCR[J].Drug Alcohol Depend,2012,124(3):223-228.
11.Zhang X,Shao S,Geng H,et al.Expression profiles of six circulating micro RNAs critical to atherosclerosis in patients with subclinical hypothyroidism:a clinical study[J].J Clin Endocrinol Metab,2014,99(5):E766-E774.
12.闫继红,杨姝,孙海梅,等.共沉默mi R-221-3p/222-3p表达抑制骨髓间充质干细胞增殖及促进软骨分化[J].中国组织工程研究,2015,15(50):8 056-8 061.
13.Wang YR,Ma ZL,Kan PC,et al.The diagnostic value of serum mi RNA-221-3p,mi RNA-382-5p,and mi RNA-4271 in ischemic stroke[J].Journal of Stroke&Cerebrovascular Diseases,2017,26(5):1 055-1 060.
14.Shen J,Hu Q,Schrauder M,et al.Circulating mi R-148b and mi R-133a as biomarkers for breast cancer detection[J].Oncotarget,2014,5(14):5 284-5 294.
15.Cheng Y,Zhang X,Li Z,et al.Highly sensitive determination of micro RNA using target-primed and branched rolling-circle amplification[J].Angew Chem Int Ed Engl,2009,48(18):3 268-3 272.