Ghawana Sanjay, Paul Asosii, Kumar Hitesh, Kumar Arun, Singh Harsharan, Bhardwaj Pardeep K, Rani Arti, Singh Ravi S, Raizada Jyoti, Singh Kashmir, Kumar Sanjay
Biotechnology Division, Institute of Himalayan Bioresource Technology (CSIR), Palampur-176 061, Himachal Pradesh, India.
BMC Res Notes. 2011 Mar 28;4:85. doi: 10.1186/1756-0500-4-85.
Secondary metabolites are reported to interfere with the isolation of RNA particularly with the recipes that use guanidinium-based salt. Such interference was observed in isolation of RNA with medicinal plants rheum (Rheum australe) and arnebia (Arnebia euchroma). A rapid and less cumbersome system for isolation of RNA was essential to facilitate any study related to gene expression.
An RNA isolation system free of guanidinium salt was developed that successfully isolated RNA from rheum and arnebia. The method took about 45 min and was successfully evaluated on twenty one tissues with varied secondary metabolites. The A260/280 ratio ranged between 1.8 - 2.0 with distinct 28 S and 18 S rRNA bands visible on a formaldehyde-agarose gel.
The present manuscript describes a rapid protocol for isolation of RNA, which works well with all the tissues examined so far. The remarkable feature was the success in isolation of RNA with those tissues, wherein the most commonly used methods failed. Isolated RNA was amenable to downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), differential display (DD), suppression subtractive hybridization (SSH) library construction, and northern hybridization.
据报道，次生代谢产物会干扰RNA的分离，特别是在使用基于胍盐的方法时。在用药用植物大黄（Rheum australe）和新疆紫草（Arnebia euchroma）分离RNA的过程中观察到了这种干扰。一个快速且不繁琐的RNA分离系统对于促进任何与基因表达相关的研究至关重要。
开发了一种不含胍盐的RNA分离系统，该系统成功地从大黄和新疆紫草中分离出了RNA。该方法耗时约45分钟，并在21种含有不同次生代谢产物的组织上成功进行了评估。A260/280比值在1.8至2.0之间，在甲醛-琼脂糖凝胶上可见清晰的28S和18S rRNA条带。
本论文描述了一种快速的RNA分离方案，该方案对迄今为止所检测的所有组织都有效。其显著特点是能够成功地从那些常用方法失败的组织中分离出RNA。分离得到的RNA适用于下游应用，如逆转录-聚合酶链反应（RT-PCR）、差异显示（DD）、抑制性消减杂交（SSH）文库构建和Northern杂交。
