Computational protocol: Interplay of light and temperature during the in planta modulation of C4 phosphoenolpyruvate carboxylase from the leaves of Amaranthus hypochondriacus L.: diurnal and seasonal effects manifested at molecular levels

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Protocol publication

[…] Total RNA was isolated by using the single-step guanidine isothiocyanate method () with the TRI® reagent (Sigma) according to the manufacturer's instructions. The RNA concentration was estimated spectrophotometrically and the quality was determined by 1.0% denatured agarose–formaldehyde gel electrophoresis.About 5 μg of total RNA was reverse transcribed to cDNA using a SuperScript® first-strand synthesis kit (Invitrogen). PCR was then performed by using Platinum® Taq DNA polymerase High Fidelity (Invitrogen). The PEPC and ribulose-1,5-bisphosphate carboxylase oxygenase small subunit (RuBisCO ssu) gene-specific primers were designed using the software, Primer3 input [Whitehead Institute for Biomedical Research (); version 0.4.0].The sequences of the primers used were as follows: PEPC, forward 5′-TATGGCTAGTGGGAAAGGTG-3′, reverse 5′-TTGCTTAACCGGGTGTTCTGC-3′; RuBisCO ssu, forward 5′-TGTAAGCAATGAAACTGAG-3′, reverse 5′-AATGGCTTCCTCTATGCTTT-3′.The PCR products were separated on 1.0% agarose gels. After confirmation of amplification and determination of the amplicon size, the products were purified by using a QIAquick® gel extraction kit (Qiagen). All procedures used were according to kit protocols. [...] All assays were performed three times for each sample. The average values ±SE are presented. Statistical analysis of the data was done using the software Sigmaplot (version 10.0). Correlation coefficients were calculated and curve fitting of data sets was done by using Microsoft Office Excel, 2007. […]

Pipeline specifications

Software tools Primer3, SigmaPlot
Applications Miscellaneous, qPCR
Organisms Amaranthus hypochondriacus
Chemicals Glucose-6-Phosphate