Semi-Quantitative PCR

The transcriptome analysis results were further validated via semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) with two types of samples: (i) the same RNA samples that were used for RNA-seq analysis and (ii) newly isolated RNA samples from independent infestation. About 3 μg of RNA was used for first-strand cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad, USA) following the manufacturer’s guidelines. Gene-specific primers for the RT-PCR were designed using Primer3 Software (https://bioinfo.ut.ee/primer3-0.4.0) (Supplementay Table S3). The PCR mix contained 30 ng of cDNA, 0.5 μM primers (forward and reverse each), 200 μM each of the dNTPs, 1 Unit of Taq polymerase and Taq buffer (Bangalore Genei Pvt. Ltd., India). The optimum PCR conditions including cycle number and cDNA amounts were standardized for each gene separately. The PCR, products were run on 1.5% agarose gel at 90 V for 1 h. and the agarose gels were documented using the Alpha Imager EP system (Cell Biosciences, USA). The captured images were analyzed using ImageJ software (Schneider et al. 2012). Twenty genes were analyzed (Fig. ​(Fig.7)7) with rice ubiquitin (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AK059694","term_id":"32969712","term_text":"AK059694"}}AK059694) as a reference gene for normalization and the fold change values were calculated between the relative expression values (REVs) of infested and control plants. One-way ANOVA was performed on fold change value data for each gene against specific planthopper and time point and means were separated by HSD following Tukey and Kramer method (Tukey 1953) on MS Excel (https://www.youtube.com/watch?v=N7mkI8_xxc4&feature=emb_logo). Negative values were expressed as decimal fraction for analysis. Results are expressed as a graph constructed based on the log (2) values of the fold change using MS Excel (Supplementary Table S4, Fig. ​Fig.77).