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Not AvailableBacterial blight is a widespread disease in pomegranate that causes great loss to farmers. The causative agent for this disease is Xanthomonas axonopodis pv. punicae. The present study was taken up to standardize protocol for quality total RNA extraction from various tissues of pomegranate, cDNA synthesis and qPCR validation of differentially expressed gene(s) identified from RNA sequencing data of susceptible and moderately resistant pomegranate genotypes upon challenge inoculation using qPCR. In the study, Phenol-Chloroform, Modified CTAB-LiCl and Trizol methods were evaluated for their efficiency to extract quality total RNA from infected and uninfected leaf and fruit tissues of pomegranate genotypes (Bhagwa and IC-1181). The concentration of extracted total RNA were quantified using Qubit Fluorometer and Qiagen QIAxpert and the quality of 18 and 28S bands of ribosomal RNA also assessed on agarose gel electrophoresis. Phenol-Chloroform method gave the highest concentration of total RNA having Qubit Fluorometer and QIAxpert readings ranged from 3.86 to 5.78 ng/μl and 543.5 to 1684.3 ng/μl, respectively. The time consumed and cost incurred on total RNA isolation were also least in Phenol-Chloroform method (50 minutes and Rs. 29.75/sample, respectively) as compared to other methods. From the high quantity total RNA, cDNA were synthesized using cDNA synthesis kit (HiMedia cDNA synthesis kit) and Xyloglucan endotransglucosylase coding gene was validated using qPCR. The qRT-PCR results showed that gene which code for Xyloglucan endo transglycosylase was slightly over expressed in the infected leaf samples of Bhagwa at infection stage 1 and 3 as compared with the control sample whereas the same gene had under expression in infected leaf sample of IC 1181 as compared to control.Not Availabl