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Quality RNA isolation, cDNA synthesis and qPCR validation of differentially expressed gene in Punica granatum L. under influence of Xanthomonas axonopodis pv. punicae

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Title Quality RNA isolation, cDNA synthesis and qPCR validation of differentially expressed gene in Punica granatum L. under influence of Xanthomonas axonopodis pv. punicae
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Creator Tele AA, Banda MA, Bachake SS, Jadhav VB, Deshpande PP, Adki VS, Gopika MK, Shinde NA, Sharma J, Parashuram S, Sangnure VR, Mundiwadikar DM and Singh NV
 
Subject Pomegranate, total RNA, Xanthomonas axonopodis pv. punicae, qPCR
 
Description Not Available
Bacterial 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.
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Date 2020-09-08T06:14:33Z
2020-09-08T06:14:33Z
2019-04-08
 
Type Research Paper
 
Identifier Not Available
2349-8234
http://krishi.icar.gov.in/jspui/handle/123456789/41047
 
Language English
 
Relation Not Available;
 
Publisher Journal of Pharmacognosy and Phytochemistry