Normalization strategy is critical for the outcome of miRNA expression analyses in the rat heart
Normalization strategy is critical for the outcome of miRNA expression analyses in the rat heart
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DOI：
10.1152/physiolgenomics.00131.2010
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发表时间：
2011-05-01
影响因子：
4.6
通讯作者：
Jonassen, Anne K.
中科院分区：
文献类型：
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作者：
Brattelid, Trond;Aarnes, Eva-Katrine;Jonassen, Anne K.
Brattelid T，Aarnes EK，Helgeland E，Guva g S，Eichele H，Jonassen AK.标准化策略对于大鼠心脏中miRNA表达分析的结果至关重要。Physiol Genomics 43：604-610，2011.首次发表于2010年12月21日; doi：10.1152/physiolgenomics.00131.2010.-由于标准化策略在进行定量PCR（qPCR）分析时对于获得可靠结果起着关键作用，因此本研究调查了几种miRNA标准化候选物在缺血再灌注离体大鼠心脏中作为标准化标准物的效率，特别参考了miRNA miR-1和miR-101 b的调节。还研究了在一个逆转录（RT）反应中包括几种miRNA的引物的可能性。Langendorff灌注的大鼠心脏进行30分钟的区域缺血和0，1，5，15，或120分钟的再灌注。分离总RNA并逆转录用于miRNA qPCR分析。通过Normalization和geNorm算法评估归一化候选物，并获得以下稳定性表达等级顺序：sno 202 <U6 B < U87 < snoRNA < 4.5S RNA A < Y1 < 4.5S RNA B < GAPDH。应用U6 B作为标准化物，发现miR-1和miR-101 b在缺血再灌注心肌中下调。此外，在TaqMan MicroRNA测定方案中，通过用两组补充引物代替无RNA酶的水，可以在一个RT反应中包括多达三个引物。这项研究表明，在通过qPCR进行miRNA表达分析时，验证标准化标准的重要性，以及miR-1和miR-101 b可能在缺血大鼠心脏的早期再灌注过程中发挥重要作用。
Brattelid T, Aarnes EK, Helgeland E, Guva g S, Eichele H, Jonassen AK. Normalization strategy is critical for the outcome of miRNA expression analyses in the rat heart. Physiol Genomics 43: 604-610, 2011. First published December 21, 2010; doi:10.1152/physiolgenomics.00131.2010.-Since normalization strategies plays a pivotal role for obtaining reliable results when performing quantitative PCR (qPCR) analyses, this study investigated several miRNA normalization candidates in regards to their efficiency as normalization standards in the ischemic reperfused ex vivo rat heart, with special reference to regulation of the miRNAs miR-1 and miR-101b. The possibility of including primers for several miRNAs in one reverse transcription (RT) reaction was also investigated. Langendorff perfused rat hearts were subjected to 30 min regional ischemia and 0, 1, 5, 15, or 120 min reperfusion. Total RNA was isolated and reverse transcribed for miRNA qPCR analysis. Normalization candidates were evaluated by the NormFinder and geNorm algorithms and the following stability expression rank order was obtained: sno202 < U6B < U87 < snoRNA < 4.5S RNA A < Y1 < 4.5S RNA B < GAPDH. Applying U6B as a normalizer it was found that miR-1 and miR-101b was downregulated in the ischemic reperfused myocardium. Furthermore, up to three primers could be included in one RT reaction by replacing RNase-free water with two supplemental sets of primers in the TaqMan MicroRNA assay protocol. This study demonstrates the importance of validating normalization standards when performing miRNA expression analyses by qPCR, and that miR-1 and miR-101b may play an important role during early reperfusion of the ischemic rat heart.