A novel method for constructing pathogen-regulated small RNA cDNA library |
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Authors: | Chuan Bao Sun Xian Ming Du |
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Institution: | a National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China b Faculty of Medicine, The University of New South Wales, Sydney, NSW 2052, Australia |
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Abstract: | 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. |
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Keywords: | Small non-coding RNA cDNA/RNA subtractive hybridization Stem-loop primer |
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