Optimizing miR-29 measurements in biobanked, heparinized samples.
Optimizing miR-29 measurements in biobanked, heparinized samples.
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优化生物库肝素化样品中的 miR-29 测量。
DOI：
10.1016/j.lfs.2019.116894
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发表时间：
2019
期刊：
影响因子：
6.1
通讯作者：
Rogers,LynetteK
中科院分区：
文献类型：
--
作者：
Warnement,CatherineM;Cismowski,MaryJ;Rogers,LynetteK
目标MicroRNA（miRs）及其在发育、正常生理和疾病中的重要性已越来越被人们所认识。我们的实验室对miR-29及其对肺发育的影响感兴趣。这些研究确定了最佳条件下肝素化，生物库样品中的miR-29的测量和比较异构体的表达patterns.Materials和MethodsThe效率的三种不同的肝素酶进行了测试，使用逆转录酶聚合酶链反应（RT-PCR）：recombinantF。肝素酶I;重组P.肝素酶II;重组P.肝素酶III;和肝素酶I（B.衍生的）。冻融的影响，以及不同的miR-29亚型的相对表达也使用RT-PCR进行了评估。肝素）和2（重组P.肝素）有效地中和肝素活性并防止对PCR的干扰。此外，单次冻融不影响miR-29- 3 p的测量，但多次冻融循环降低了可测量的miR水平。最后，-3p链在人和小鼠血浆中的所有三种亚型中表达最丰富。显著性我们的发现说明，需要针对特定的miR和待测样品类型优化特定条件。
AimsMicroRNAs (miRs) and their importance in development, normal physiology, and disease have become increasingly recognized. Our laboratory is interested in miR-29 and its effects on lung development. These studies set out to identify optimal conditions for the measurement of miR-29 in heparinized, biobanked samples and to compare isoform expression patterns.Materials and methodsThe efficiency of three distinct heparinases were tested using reverse transcriptase polymerase chain reaction (RT-PCR): recombinantF. Heparinumheparinase I; recombinantP. heparinusheparinase II; recombinantP. heparinusheparinase III; and heparinase I (B. efferthii-derived). The effects of freeze/thaws, and the relative expression of different miR-29 isoforms were also assessed using RT-PCR.Key findingsOur investigations determined that heparinase 1 (recombinantF. Heparinum) and 2 (recombinantP. heparinus) at 1 or 2 h incubation efficiently neutralized heparin activity and prevented interference with the PCR. Also, a single freeze/thaw did not affect the measurement of miR-29-3p but multiple freeze/thaw cycles decreased the measureable miR levels. Finally, the -3p strand was most abundantly expressed in all three isoforms in both human and mouse plasma.SignificanceOur findings illustrate that specific conditions need to be optimized for the particular miR and the type of sample being tested.
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影响因子：
2.5
作者：
E. Flowers;E. Froelicher;B. Aouizerat
通讯作者：
E. Flowers;E. Froelicher;B. Aouizerat
microRNA 测定方法的阴阳。
DOI：
10.2174/2211536605666160725130028
发表时间：
2017
期刊：
MicroRNA
影响因子：
--
作者：
Audrey Guttin;Hélène Ipas;M. Barbado;C. Mouret;E. Garçion;J. Issartel
通讯作者：
J. Issartel
影响因子：
4.8
作者：
Kroh, Evan M.;Parkin, Rachael K.;Mitchell, Patrick S.;Tewari, Muneesh
通讯作者：
Tewari, Muneesh
影响因子：
3.8
作者：
Kim D;Chang HR;Baek D
通讯作者：
Baek D
影响因子：
8.3
作者：
Rogers, Lynette K.;Robbins, Mary;Tipple, Trent E.
通讯作者：
Tipple, Trent E.