2.4. RNA Extraction by using Gene JET Plant RNA Purification Minikit
Leaf samples were collected from each replication and immediately transferred to liquid nitrogen. Put 100 mg of plant tissue into liquid nitrogen and grind thoroughly with a mortar and pestle. after grinding the tissue powder were quickly transferred into 1.5 ml micro centrifuge tube which Containing 500 μL of Plant RNA Lysis Solution and Vortex all the mixture for 15-20 seconds to mix thoroughly and after that Incubate the samples for 3 min at 56°C and then Centrifuge for 5 min at ≥20,000 × g (≥14,000 rpm). Collect the supernatant 550 μL and transfer to the clean micro centrifuge tube and then Add 250 μL of 96% ethanol and Mix by pipetting and then transferred the mixture into purification column inserted in a collection tube. Centrifuge the column for 1 min at 12,000 × g (~11,000 rpm). Discard the flow through solution and reassemble column and collection tube. Then we Add 700 μL of Wash Buffer WB 1 to the purification column (ethanol has been added to Wash Buffer WB 1). Centrifuge for 1 min at 12,000 × g (~11,000 rpm). Discard the flow-through and collection tube. Place the purification column into a clean 2 mL collection tube. Then Add 500 μL of Wash Buffer 2 to the purification column (ethanol has been added to Wash Buffer 2). Centrifuge for 1 min at 12,000 × g (~11,000 rpm). Discard the flow-through solution and reassemble column and collection tube after that we repeat this step again. To elute the RNA, add 50 μL of nuclease-free water to the centre of the purification column membrane and centrifuge for 1 min at 12,000 × g (~11,000 rpm). Discard the purification column and extracted RNA immediately stored at -20°C for further analysis.