title: Two modified RNA extraction methods compatible with transcript profiling and gene expression analysis for cotton roots.
abstract: Efficient isolation of high-quality RNA is of prime importance for optimal transcript profiling results and further gene expression analysis. However, it is difficult for cotton roots because of lower-than-average RNA content and high content of polysaccharides, polyphenols, and other secondary metabolites. To develop simple and reliable protocols for high-quality RNA extraction from cotton roots for transcript profiling and gene expression analysis, some modifications were introduced to a reported plant RNA isolation protocol and a reagent kit method. Using method A, we successfully extracted high-quality RNA for transcript profiling from cotton roots. Gel electrophoresis analysis and polymerase chain reaction (PCR) assay indicated that RNA had good integrity without protein and genomic DNA contamination. Furthermore, the A260/280 (1.9) and A260/230 (1.6) ratios indicated that the isolated RNA was of high purity. Using method B, about 7 µg total RNA of high quality could be obtained from 0.1 g samples from cotton roots, which can be used for reverse-transcription (RT)-PCR and quantitative real-time RT-PCR. The two RNA extraction methods were used to investigate different gene expression of cotton roots (Gossypium hirsutum) infected by weak pathogenic Verticillium dahliae and the results showed they can satisfy the transcript profiling and quantitative real-time RT-PCR requirements for RNA. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.
authors.affiliation: College of Life Science, Chongqing Normal University, Chongqing, China.
authors.forename: Chengjian
authors.initials: C
authors.lastname: Xie
authors.forename: Chunyan
authors.initials: C
authors.lastname: Wang
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Wang
authors.forename: Xingyong
authors.initials: X
authors.lastname: Yang
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D003368:Gossypium / Q000235:genetics* / Q000382:microbiology; D010840:Phytosterols / Q000235:genetics / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology; D018517:Plant Roots / Q000235:genetics*; D020871:RNA Stability; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D020172:Verticillium / Q000472:pathogenicity
chemical_list: D010840:Phytosterols; D018749:RNA, Plant