2.3.3 RNA extraction and quantitative real-time PCR (qRT-PCR) reaction
Root samples (1 g) of tomato plants were fine powdered with the help of pre-sterilized and pre-chilled mortar and pestle using liquid nitrogen. An aliquot of macerated tissue (100 mg per sample) were used for RNA extraction. An extraction of RNA was processed using RNA-easy Plant Mini Kit (Qiagen, Germany), according to the manufacturer’s instructions. RNA quality was quantified using a Nano-drop spectrophotometer. QuantiTect Reverse Transcription Kit (Qiagen, Germany) with random hexamers were used for cDNA synthesis from 1 μg of total RNA. PCR mixtures (20-μl final volume) were prepared which contained RNAse free water, 0.2μM each of forward and reverse primers, 1.5μl cDNA template and 10 μlSYBR1. ThePCR programme used was of initial denaturation at 95˚C (10 min); 40 cycles of- denaturation (95˚C for 30 sec), annealing (58˚C for 30 sec) extension (72˚C, 30 sec) and final extension 72˚C for 1 min). QRT-PCR were performed in triplicate using a Bio-Rad iQ5 Multi-coloured real time PCR detection system. Actin gene was used as reference gene due to its consistent expression in tomato which does not vary after the nematode infections. Genes and respective primers used in the study are given in Table 1. Relative fold changes of genes expression were calculated by the 2-ΔΔCT method (Livak andSchmittgen 2001).