Total Plant RNA Isolation-Trizol Reagent Method

• Total Plant RNA Isolation Using Trizol Reagent

1. Crush 100 mg of tissues in 1mL of triazole reagent by pestle mortar or micro pestle 1.5 or 2.0 mL RNase free Eppendorf tubes.
*Incubate for 2-3 minutes at room temperature.
2. Add 200 uL chloroform and mix by gently inverting the tube.
*Incubate for 2-3 minutes at room temperature.
3. Centrifuge at 12000xg at 4oC for 15 minutes.
4. Transfer the supernatant into fresh 1.5 mL RNA’s free tube.
5. Add 500 μL isopropanol mix gently by inverting the tube.
*Incubate for 10 minutes at room temperature.
6. Centrifuge at 12000xg at 4oC for 10 minutes.
*Discard the supernatant and keep the pallet in the tube.
7. Add 1mL of ice-chilled 75% ethanol to wash the pallet.
8. Centrifuge at 7500xg at 4oC for 5 minutes.
*Discard the supernatant and keep the pallet in the tube.
*Keep lid opened tube at 4oC for 15 min to air dry the pallet.
9. Add 50-100 uL RNA’s free water in pallet and mix by gentle pipetting.
10. Incubate for 10-15 minutes at 50-60 oC temperature.
11. Use fresh RNA in RT-PCR or Store at -80oC for future use.
*cDNA Synthesis (20 uL Rxn) Protocol by using Thermo Scientific

• RevertAid First Strand cDNA Synthesis Kit #K1622

1. Add the following reagents into a sterile, nuclease-free tube on ice in Template RNA 4 µL
*Reverse primer (O.dT18 or hexamer primer) 1 µL Nuclease free water 7 µL
2. Incubate at 65°C for 5 minutes.
3. Add the following components in the indicated order 5X Reaction Buffer 4 µL
*RiboLock RNase Inhibitor (20 U/µL) 1 µL 10 mM dNTP Mix 2 µL
*RevertAid M-MuLV RT (200 U/µL) 1 µL Mix gently and centrifuge briefly.
4. Incubate For oligo(dT)18 or gene-specific primed cDNA synthesis,
*Incubate for 60 min at 42°C.
*For random hexamer primed synthesis
*Incubate for 5 min at 25°C followed by 60 min at 42°C.
5. Terminate the reaction by heating at 70°C for 5 min.
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• Preparation of a 25 µL PCR reaction using ThermoScientific

1. DreamTaq Green PCR Master Mix (2X)
2. DreamTaq Green PCR Master Mix (2X) 12.5 µL
3. Forward primer 1 µL
4. Reverse primer 1 µL
5. Template DNA 4 µL
6. Water, nuclease-free 6.5 µL
7. Total volume 25 µL

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Written By:

Dr. Adnan Ahmed

Tahir Mahmood

Department of Plant Pathology-Agriculture

The Islamia University of Bahwalpur-Pakistan.