A novel method for constructing pathogen-regulated small RNA cDNA library

作者：Sun, Chuan Bao^*; Du, Xian Ming; He, Yu Ke

来源：Biochemical and Biophysical Research Communications, 2010, 397(3): 532-536.

DOI：10.1016/j.bbrc.2010.05.151

• Small non-coding RNA
• cDNA/RNA subtractive hybridization
• Stem-loop primer

Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp. carotovora (Ecc), often leads to soft rot disease in the plant Brassica campestris L ssp. pekinensis (Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem-loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 3' end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing.

• 出版日期2010-7-2
• 单位中国科学院; 中国科学院上海生命科学研究院