Computational protocol: Short-Term Complete Submergence of Rice at the Tillering Stage Increases Yield

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Protocol publication

[…] Total RNA was extracted from roots with the TransZol reagent (Invitrogen, Carlsbad, CA, USA). DNase I (Invitrogen, Carlsbad, CA, USA) was added to eliminate contaminating genomic DNA. The RNA was reverse transcribed into cDNA with the iScript cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Using this cDNA as a template, the expression of OS-ACS3 was determined by real-time polymerase chain reaction (qPCR). The PCR reaction conditions were as follows: 95°C for 30 s, followed by 40 cycles of 94°C for 10 s, 25°C for 25 s and 72°C for 30 s, with a final step of 72°C for 7 min. The experiment was repeated three times. Negative controls were set up with ddH2O and RNA in place of the cDNA template to determine whether exogenous DNA or genomic DNA contamination was present.The primers used in the experiments were designed using Primer Express Software (Foster City, CA, USA) and synthetized by Shanghai Bio Asia Biotechnology Co., Ltd. The purity of the primers was greater than 99%, and Actin was used as the reference gene. The forward primer designed to amplify OsActin (NCBI GenBank: NM_001058705) was 5’-CCAAGGCCAATCGTGAGAAGA-3’, and the reverse primer was 5’- AATCAGTGAGATCACGCCCAG-3’. The primer’s specificity was determined using BL0AST and melting curve analysis. The forward primer designed to amplify OS-ACS3 (NCBI GenBank: AC135427) was 5’-AGTGTGGGAGAGGGTGTGAC-3’, and the reverse primer was 5’-GGTGGAAGTGCCAACAAAG-3’. The expected product size was 214 bp.Tenfold dilutions of cDNA templates from plants treated with submergence were used for gradient PCR of OS-ACS3 and Actin to obtain standard curves and determine amplification efficiencies. Relative quantitative analysis of each sample was performed according to the literature []. [...] All data were processed with Excel, and an analysis of variance (ANOVA) was performed using SPSS software (SPSS Inc., Chicago, IL, USA). T-tests were employed for comparisons between groups. Charts were drawn with SigmaPlot. Differences at P ≤ 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools Primer Express, SPSS, SigmaPlot
Applications Miscellaneous, qPCR
Organisms Oryza sativa