Gauthier E R, Madison S D, Michel R N
Department of Chemistry and Biochemistry, Laurentian University, Ramsey Lake Road, Sudbury, Ontario, Canada P3E 2C6.
Pflugers Arch. 1997 Mar;433(5):664-8. doi: 10.1007/s004240050328.
We describe an adapted version of the Chomczynski and Sacchi [Anal Biochem (1987) 162:156-159] RNA isolation procedure that can be performed in less than 1 h on small (<15 mg) tissue samples, using commonly available reagents. Our modifications included: (1) one rather than two precipitation steps in the aqueous phase with 99% ethanol and (2) elimination of the 1-h incubation step at -20 degrees C. Our adaptations resulted in RNA yield (microg/mg of tissue) and purity (260/280 nm ratios) comparable to those of the original procedure. Furthermore, the isolated RNA was successfully utilized in reverse transcriptase-polymerase chain reaction assays, suggesting that it was essentially free of carry-over contaminants that could inhibit enzymatic reactions. When tested on tissue sample sizes of 7-12 mg, our adapted procedure allowed the recovery of enough total RNA for use in techniques such as Northern blot analysis. Our modified technique is therefore an inexpensive alternative to commercially available kits when isolating good-quality RNA from very small tissue samples, such as those obtained from needle biopsies.
我们描述了一种对Chomczynski和Sacchi [《分析生物化学》(1987年) 162:156 - 159] RNA分离方法的改良版本，该方法使用常见试剂，可在不到1小时内对小（<15毫克）组织样本进行操作。我们的改进包括：(1) 在水相中用99%乙醇进行一步而非两步沉淀；(2) 省去了在-20℃下1小时的孵育步骤。我们的改良使RNA产量（微克/毫克组织）和纯度（260/280纳米比值）与原方法相当。此外，分离得到的RNA成功用于逆转录-聚合酶链反应分析，表明其基本不含可能抑制酶促反应的残留污染物。当对7 - 12毫克的组织样本进行测试时，我们的改良方法能回收足够用于Northern印迹分析等技术的总RNA。因此，在从非常小的组织样本（如针吸活检获得的样本）中分离高质量RNA时，我们的改良技术是市售试剂盒的一种廉价替代方法。
