• Extraction of RNA using standard method:

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    RNA Isolation should be done in clean RNase free working zone. Fresh Plant leaf tissue is used for isolation of RNA which is collected in liquid nitrogen container to avoid degradation of RNA.

  • Steps involved for isolation of RNA from leaf tissue are as follows:

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    1. Plant leaf tissue (100 mg) are ground to fine powder in liquid nitrogen using autoclaved mortar and pestle and homogenized in 1 ml Trizol reagent. (TRIzol Reagent is a ready to use mixture of phenol, guanidine isothiocyanate, red dye).
    2. The homogenate is incubated at room temperature for 5 min. Samples are mixed with 200 µl chloroform, incubated at room temperature for 2 min and centrifuged at 12,000 rpm for 15 min at 4° C.

    The aqueous phase is transferred to a fresh tube and RNA is precipitated by adding 0.5 ml isopropyl alcohol. RNA pellet obtained after centrifugation is washed with 70% ethanol. The pellet is resuspended in 20 to 100µl RNase free sterile water.

    The RNeasy Plant Mini Kit provides lysis buffers solutions, Buffer RLT is the lysis buffer of choice but Buffer RLT can cause solidification of some samples, due to secondary metabolites in the tissue. Buffer RLC should be used in such samples. Add either 10 μl β-mercaptoethanol (β-ME), or 20 μl 2 M dithiothreitol (DTT)*, to 1 ml Buffer RLT or Buffer RLC before use. Buffers with DTT or β-ME can be stored at room temperature for up to 1 month. Add 4 volumes of ethanol (96–100%) to Buffer RPE for a working solution.

    1. Plant leaf tissue (100 mg) is ground to fine powder in liquid with mortar and pestle. after thorough grinding, decant tissue powder and liquid nitrogen into RNase-free, liquid-nitrogen–cooled 2 ml microcentrifuge tube. Allow the liquid nitrogen to evaporate, but do not allow the tissue to thaw.
    2. Add 450 μl Buffer RLT or Buffer RLC to a maximum of 100 mg tissue powder, Vortex vigorously.
    3. Transfer the lysate to a QIAshredder spin column (lilac) placed in a 2 ml collection tube. Centrifuge for 2 min at full speed. Transfer the supernatant of the flow-through to a new microcentrifuge tube without disturbing the cell-debris pellet.
    4. Add 0.5 volume of ethanol (96–100%) to the cleared lysate, and mix immediately by pipetting. Do not centrifuge. Proceed immediately to step 5. Transfer the sample (usually 650 μl), with any precipitate, to an RNeasy Mini spin column (pink) in a 2 ml collection tube. Close the lid, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow through.
    5. Add 700 μl Buffer RW1 to the RNeasy spin column. Close the lid, and centrifuge for 15 s at ≥8000 x g. Discard the flow-through.
    6. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 15 s at ≥8000 x g. Discard the flow-through.
    7. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 2 min at ≥8000 x g. (Optional: Place the RNeasy spin column in a new 2 ml collection tube. Centrifuge at full speed for 1 min to dry the membrane).
    8. Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30–50 μl RNase-free water directly to the spin column membrane. Close the lid, and centrifuge for 1 min at ≥8000 x g to elute the RNA.
    9. If the expected RNA yield is >30 μg, repeat step 9 using another 30–50 μl of RNase-free water. Alternatively, use the elute from step 9 (if high RNA concentration is required). Reuse the collection tube from step 9.