Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.
Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses.
Changxun
C
Fang
Tiecheng
T
Xu
Changliang
C
Ye
Likun
L
Huang
Qingshui
Q
Wang
Wenxiong
W
Lin
D011232:Chemical Precipitation; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D015723:Gene Library; D006812:Humic Substances; D008828:Microbiological Techniques / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D010944:Plants; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D058441:Rhizosphere; D012988:Soil Microbiology; D013048:Specimen Handling / Q000379:methods*
D018076:DNA, Complementary; D006812:Humic Substances; D012313:RNA