Computational protocol: Transcription of putative tonoplast transporters in response to glyphosate and paraquat stress in Conyza bonariensis and Conyza canadensis and selection of reference genes for qRT-PCR

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Protocol publication

[…] Eight genes commonly used as qRT-PCR reference genes in Arabidopsis thaliana were evaluated as candidate reference genes in C. bonariensis and C. canadensis []. The Arabidopsis thaliana nucleotide coding sequences of candidate reference genes were used as queries in a BLASTN search optimized for discontinuous megablast against the Conyza canadensis genome [] from the NCBI assembly database []. The gene names, description, and details of the BLAST search are presented in .Homolog detection was performed using a reciprocal BLAST search as described previously []. The C. canadensis genomic sequences most similar to the A. thaliana reference coding sequences were used to design qRT-PCR primers. Primer design was performed using the Primer-BLAST tool based on Primer 3 [, ]. The primers used for the ACTIN genes were previously published by Peng et al. []. The primers for EPSPS were designed to amplify conserved regions of all three known copies of the EPSPS gene from C. canadensis []. New primers were designed for M10 and M11 using as templates the direct sequences of the cDNA regions amplified by the primers originally developed by Peng et al. [] (). The primers designed used to amplify cDNA fragment were: M10 Forward (5’-TTGGCTCAACTTCGTGGTATCGGG-3′) and reverse (5′-CCAAGAAATTCCAAGCGGAACCCT-3′) with an amplified fragment of 253 bp; M11 Forward (5′-ATGCTGTCTTCTTTTACCTTTGC-3′) and reverse (5′-CGACTTCCCACTACCAGTTCTTC-3′) with an amplified fragment of 393 bp [].The thermocycle conditions were as follows: one initial cycle at 95°C for 2 min, followed by 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 2 minutes. The reaction was terminated with an extension cycle at 72°C for 7 minutes.Primer pairs were selected based on low predicted likelihood of forming secondary structures (including self-dimerization, hairpin, and self-annealing), a prediction made by the OligoCalc online tool []. Primer specificities were tested by performing PCR using cDNA of both Conyza spp. as templates and running the amplified product on a 2% agarose gel. We also analyzed the shape of the dissociation curve to confirm each product had a single peak melting curve (). Amplification efficiencies were calculated using the slope of Ct values derived from a four-fold cDNA dilution series. Efficiencies were calculated using the formula: efficiency (%) = (10(-1/slope)-1)*100 and ranged from 84 to 99% (). [...] The cDNA samples from three biological replicates of each line and treatment combination were selected to analyze the stability of the candidate genes’ transcript levels across species, genotypes and herbicide treatments. Selection of candidate genes was performed independently for each herbicide and species. Evaluation of candidate genes was based on the number of cycles required for the fluorescence signal to surpass the threshold level, or cycle threshold (Ct). Ct values were assigned by a built-in algorithm in the qRT-PCR system using default settings. Ct values were assessed visually and no manual adjustments were necessary. Presence of outliers was tested using the Grubb’s test (p<0.05), and outliers were removed prior to further analysis []. Ct means and standard deviations for evaluated reference genes are available in .Many algorithms are available to evaluate gene stability, and the final outcome of gene stability is comparable among many of these algorithms []. Candidate gene stability was evaluated using the NormFinder algorithm because this algorithm can evaluate different sample groups and separate intra- versus inter-group variation []. The variation is summarized in a single stability parameter with smaller values indicating greater transcription stability. NormFinder stability parameters were calculated for each treatment and species and genes were ranked based on stability. A consensus ranking was calculated using the Monte Carlo algorithm based on the Spearman’s distances across rankings performed by RankAggreg package in R []. The three most stable genes were selected as reference genes to assay the transcript levels of target genes. […]

Pipeline specifications

Software tools BLASTN, Primer-BLAST, OligoCalc, NormFinder, RankAggreg
Applications Gene expression microarray analysis, qPCR
Diseases Agricultural Workers' Diseases
Chemicals Amino Acids, Paraquat