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.