Total RNA Isolation Kit (Plant)

The Total RNA Isolation Kit provides a fast, simple, and cost-effective method for isolation of total RNA from plant samples. Detergents and chaotropic salt are used to lyse cells and inactivate RNase. The specialized high-salt buffering system allows RNA species longer than 100 bases to bind to the glass fiber matrix of the spin column. The Total RNA Isolation Kit is suitable for a variety of routine applications including RT-PCR, cDNA Synthesis, Northern Blotting, Differential display, Primer Extension and mRNA selection. The entire procedure can be completed within 60 minutes.

• Sample: 100 mg of fresh plant tissue or 25 mg of dry plant tissue
• Format: Spin column
• Operation time: within 1 hour
• Elution volume: 50-200 μl
• Yield: up to 30 μg

Kit Contents

• Delivering high-quality total RNA with the fast procedure
• Ready-to-use RNA for high performance in any downstream application
• Consistent RNA yield from the starting material with a small amount

Step 1 Sample Preparation

1. Cut off 100 mg of fresh plant tissue or 50 mg of dry plant tissue.
2. Grind the sample under liquid nitrogen to a fine powder using a mortar and pestle.

Step 2 Lysis

1. Add 1 ml of Buffer RP and 10 μl of ß-mercaptoethanol to the sample in the mortar and grind the sample until it is completely dissolved.
2. Transfer the dissolved sample to a RNase-free 1.5 ml microcentrifuge tube.
3. Incubate at 75°C for 30 minutes. (invert the tube every 10 minutes)
4. Centrifuge at 2-8°C at 14-16,000 x g for 10 minutes.
5. Transfer carefully the clear supernatant to a new 1.5 ml microcentrifuge tube.

Step 3 RNA Binding

1. Add the half volume of Isopropanol to the sample from Step 1 and shake vigorously (e.g. add 250 µl of Isopropanol to 500 µl of sample).
2. Place a Column RP in a 2 ml Collection Tube.
3. Transfer the sample mixture to the Column RP.
4. Centrifuge at 14-16,000 x g for 30 seconds.
5. Discard the flow-through and transfer the remaining mixture to the same Column RP.
6. Centrifuge at 14-16,000 x g for 30 seconds.
7. Discard the flow-through and place the Column RP back in the same Collection Tube.

Step 4 Wash

Add 400 µl of Buffer W1 into the Column RP.
Centrifuge at 14-16,000 x g for 30 seconds.
Discard the flow-through and place the Column RP back into the same Collection Tube.
Add 600 µl of Buffer W2 (ethanol added) into the Column RP.
Centrifuge at 14-16,000 x g for 30 seconds.
Discard the flow-through and place the Column RP back into the same Collection Tube.
Centrifuge at 14-16,000 x g again for 3 minutes to dry the column matrix.

Step 5 Elution

1. To elute RNA, place the Column RP in a new RNase-free 1.5 ml microcentrifuge tube.
2. Add 50-200 µl of Buffer RE to the center of each Column RP, let it stand for 2 minutes, and centrifuge at 14-16,000 x g for 2 minutes.

#Optional DNase treatments can be followed to remove the unwanted DNA residue.

• During the operation, always wear the latex or vinyl gloves while handling reagents and RNA samples to prevent the RNase contamination.
• Buffers RP and W1 contain irritants. Wear gloves when handling these buffers.
• Add 60 ml of the ethanol to the Buffer W2 before use.
• Check Buffers before use for salt precipitation. Re-dissolve any precipitate by warming up to 37°C.
• Research Use Only. Not intended for any animal or human therapeutic or diagnostic uses.