Houze T A, Gustavsson B
Ostra University Hospital, University of Göteborg, Sweden.
Biotechniques. 1996 Dec;21(6):1074-8, 1080, 1082. doi: 10.2144/96216st05.
We describe a novel approach for the isolation and quantification of mRNA from archival specimens by using oligo(dT)25 paramagnetic beads and the reverse transcription polymerase chain reaction (RT-PCR). A preliminary study is conducted to evaluate experimental conditions and the effects of varied periods of sonification on the yield of mRNA isolated from formalin-fixed, paraffin-embedded human biopsy samples. Validation of this approach will involve the comparison of observed beta-actin gene expression levels among different samples from the same patient after various periods of sonification. The novelty of this method is that it offers a means of analyzing archival samples using semiquantitative RT-PCR following sonification-enhanced mRNA extraction. The utility of this protocol allows for gene expression studies to be done effectively using the formalin-fixed, paraffin-embedded archival samples that are commonly available in all hospital pathology departments.
我们描述了一种通过使用oligo(dT)25顺磁珠和逆转录聚合酶链反应(RT-PCR)从存档标本中分离和定量mRNA的新方法。进行了一项初步研究,以评估实验条件以及不同超声处理时间对从福尔马林固定、石蜡包埋的人体活检样本中分离的mRNA产量的影响。这种方法的验证将涉及比较同一患者在不同超声处理时间后不同样本中观察到的β-肌动蛋白基因表达水平。该方法的新颖之处在于,它提供了一种在超声增强mRNA提取后使用半定量RT-PCR分析存档样本的方法。该方案的实用性使得能够使用所有医院病理科普遍可用的福尔马林固定、石蜡包埋的存档样本有效地进行基因表达研究。