Detection of stress-induced 5′tRNA halves by poly(A) tailed-RNase H digestion-RT-PCR
10.7644/j.issn.1674-9960.2015.06.012
- VernacularTitle:Poly（A）加尾-RNase H消化-RT-PCR法分析应激诱导生成5′端tRNA半分子
- Author:
Di LIU
;
Hanjiang FU
;
Jilai LIU
;
Jie ZHU
;
Xiaofei ZHENG
- Publication Type:Journal Article
- Keywords:
tRNA;
5′tRNA half;
poly(A) polymerase;
reverse transcriptase polymerase chain reaction;
endonucleas-es;
RNase H;
detection
- From:
Military Medical Sciences
2015;(6):460-463
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a simple and quick method for detection of stress-induced 5′transfer RNA( tRNA) halves.Methods Total RNA purified from stress induced cells was polyadenylated by poly( A) polymerase, and then degen-erate DNA probes were used to hybridize with 3′tRNA-halves of intact tRNAs,while RNase H specifically degraded the 3′tRNA-halves strand in tRNA-DNA probes hybrids.Using the RNase H digestion total RNA as templates, complementary DNA( cDNA) was synthesized by oligo ( dT) n-anchored primers.The primer of 5′tRNA halves and anchored-primer were used to amplify 5′tRNA halves by PCR.Results The results showed that the method of poly ( A )-tailed-RNase H digestion-RT-PCR could be successfully used to detect stress-induced 5′tRNA halves.Conclusion A simple and quick method for detection of 5′tRNA halves has been established,which is a user-friendly tool for 5′tRNA halves detection and function research.
