Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA
Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA
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DOI：
10.1515/cclm.2000.026
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发表时间：
2000-02-01
影响因子：
6.8
通讯作者：
Mitterbauer, G
中科院分区：
文献类型：
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作者：
Mannhalter, C;Koizar, D;Mitterbauer, G
逆转录聚合酶链反应（RT-PCR）分析正日益成为血液和肿瘤疾病诊断和预后评估的一部分。目前，在诊断实验室中使用不同的RNA分离方法。没有数据可用于敏感检测断点簇区域-abelson（BCR-ABL）基因转录本的适用性。我们用3种RNA分离试剂（TRI-zol（TM）、RNAzol（TM）、FastTube（TM）试剂）从4名患者（1名白细胞增多症患者、1名接受干扰素治疗的慢性粒细胞白血病（CML）患者和2名骨髓移植后的CML患者）的单核细胞（MNC）组分和裂解血液样品中提取RNA。如琼脂糖凝胶电泳和在260 nm处的分光光度测量所示，使用RNAzol（TM）的RNA产率略高于使用TRIzol（TM）的RNA产率。FastTube（TM）试剂不适合从MNC中分离RNA，未对裂解血液进行评价。ABL基因的定量竞争性RT-PCR扩增显示了用RNAzol（TM）和TRIzol（TM）分离的RNA的相当结果。在从裂解全血中提取的RNA样本中，通过多重PCR扩增4个选定的参考基因（胆色素原脱氨酶（PBGD）、ABL、跨越22号染色体上的BCR的基因和视黄酸受体α（RARA）），确认是否存在可扩增的RNA/cDNA。使用RNAzol（TM）获得了高质量的无DNA RNA，并且通过单步RT-PCR成功检测到2x 10（4）个正常细胞中的1个BCR-ABL阳性（对易位t [9; 22]具有特异性）细胞。在用TRIzol（TM）分离的RNA中，观察到基因组DNA的主要污染，这显著损害了RT-PCR分析结果的解释。
Reverse transcription polymerase chain reaction (RT-PCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRI-zol(TM), RNAzol(TM), FastTube(TM) reagent). RNA yield was slightly higher with RNAzol(TM) than with TRIzol(TM) as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube(TM) reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol(TM) and TRIzol(TM). In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol(TM), and 1 BCR-ABL-positive (specific for translocation t [9; 22]) cell among 2x10(4) normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol(TM), major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.