QS
Qingyao Shu
  • Institute of Nuclear Agricultural Sciences, Zhejiang University, China
Research fields
  • Plant science
An Efficient Procedure for Protoplast Isolation from Mesophyll Cells and Nuclear Fractionation in Rice
Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. This protocol, adapted from Zhang et al. (2011), describes a procedure for the isolation of rice protoplasts from green tissue and shows an efficient and rapid method for isolation of nuclei form these protoplasts which are commonly used in a variety of experimental procedures including the isolation of high-molecular-weight DNA (Watson and Thompson, 1986), in vitro DNA synthesis (Roman, 1980), isolation of labeled transcripts for differential screening of cDNA libraries (Somssich et al., 1989), preparation of nuclear extracts for in vitro transcription systems (Roberts and Okita, 1991), isolation of nuclear proteins (Harrison et al., 1992) and studies of protein targeting to the nucleus (Hicks and Raikhel, 1993).
Isolation of Polysome-bound mRNA from Rice Solid Tissues Amenable for RT-PCR and Profiling Experiments
Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and western blotting, genome-wide microarray analysis or qRT-PCR. Here, we describe a method for the isolation and quantification of high-quality polysome-bound mRNA complexes from small quantities of liquid-nitrogen-frozen solid tissue samples of rice shoots/roots. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level.
RNA Editing Detection by Direct Sequencing
RNA editing is a widespread post-transcriptional phenomenon through which primary RNA sequences are altered by nucleotide insertion/deletion or base conversion. It occurs in a variety of organisms and cooperates with alternative splicing in increasing both proteomic and transcriptomic complexity. We describe here a method allowing RNA editing events detection by performing direct sequencing of both genomic DNA and cDNA from the same source.
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