Computational protocol: Quantitative proteomics reveals the effect of protein glycosylation in soybean root under flooding stress

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Protocol publication

[…] A portion (100 mg) of samples was ground into powder in liquid nitrogen with a sterilized mortar and pestle. Total RNA was extracted from the tissue powder using an RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA). RNA was reverse-transcribed using an iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. qRT-PCR was performed in a 10 μL reaction assay using SsoAdvanced SYBR Green Universal Supermix (Bio-Rad) and a MyiQ single-color real-time PCR detection system (Bio-Rad). The PCR conditions were as follows: 95°C for 210 s, then 45 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Gene expression was normalized using 18S rRNA as an internal control. The primers were designed using the Primer3 web interface (http://frodo.wi.mit.edu). Specificity of primers used for the analysis was checked by the BLASTN search against the Phytozome-G.max database with the designed primers sequences as queries (Supplemental Table ) and by melting curve analysis. […]

Pipeline specifications

Software tools Primer3, BLASTN
Databases Phytozome
Application qPCR
Organisms Glycine max