- 1
cultured cell 혹은 animal tissue에서 RNA를 분리하려고 합니다.
QIAGEN kit로 RNA isolation을 할때 lysis buffer에 beta-mercaptethanol 을 넣어주라고 하는데, 그 농도가 RNA isolation에 얼마나 중요한지 궁금합니다. 적게 넣거나 더 넣으면 어떤 일이 일어나는지 궁금합니다.
- RNA isolation
각 분야 한인연구자와 현업 전문가분들의 답변을 기다립니다.
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답변
장성재님의 답변
2016-06-17- 1
FAQ
Why do I have to add beta-mercaptoethanol (beta-ME) to lysis Buffer RLT of the RNeasy Kits?
(from Qiagen website: https://www.qiagen.com/kr/resources/faq?id=eac3139e-6c6c-4172-b61f-18d61b6cdd1e)
FAQ ID -101
When working with RNA, care must be taken to avoid degradation by RNases, which are extremely stable and active. Intracellular RNases are released during the lysis step of the RNA isolation procedure and must be rapidly and thoroughly inactivated to obtain high-quality RNA.
Beta-mercaptoethanol (ß-ME) is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds and destroying the native conformation required for enzyme functionality. In combination with the strong, but temporary denaturing effects of guanidinium isothiocyanate (GITC) contained in buffer RLT of the RNeasy Kits, any RNases present in the material to be extracted from will be completely inactivated.