RNA extraction protocol

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RNA extraction protocol:

RNA extraction can be easily contaminated by materials in the air, or scarps from any part of your body, thus be careful when you process RNA extraction, otherwise it will lead to a devastating failure when you proceed to downstream experiments such as RT-PCR.

Day 1:

1. Pre-spun the Phase Lock Gel-Heavy 2ml tubes at 15,000g 1min 4°C;

2. Add 200μl Trizol reagent, mix by homogenizer, then add 400μl Trizol reagent and shake the tube by hand for 20 times (do not vortex);

3. Incubate 5min at room temperature;

4. Transfer to pre-spun Phase Lock Gel-Heavy 2ml tubes;

5. Add 120μl chloroform and shake vigorously by hand for 15 times;

6. Centrifuge 16,000g, 10min at 4°C;

7. Transfer the supernatant to a clean (RNase-free) 1.5ml tube;

8. Add 0.7-0.8 volumes of isopropanol;

9. Mix well by shaking and vortexing;

10. Precipitate overnight at -20°C.

Day 2:

1. Spin at full speed for 28min at 4°C;

2. Carefully remove the supernatant by hand;

3. Wash once with 500μl 75% ethanol;

4. Spin at full speed for 10min;

5. Spin (up to full speed and stop);

6. Remove the remaining supernatant by pipet;

7. Treatment with DNase  I: Add 88μl dH2O, then put them in pre-warmed warmer at 55ºC for 10 min; Cool them down at roomtemperature and add 10μl RDD, then add 2.5μl DNase I; Incubate at roomtemperatre for 10min;

8. Air dry for 1-2min (Do not over dry)

9. Add 88μl RNase-free water and incubate at 55°C for 10-15min;

10. Add 350μl RLT mix, then add 250μl 100%ethanol and mix well;

11. Transfer the sample (approximately 200μl) to RNase-free column tube and centrifuge 15s at 10,000rpm and discard the fluid;

12. Add 500μl RPE buffer and centrifuge 15s at 10,000rpm, then repeat once;

13. Put the column into a 2ml collection tube and centrifuge at full speed for 1min;

14. Place the RNase-free spin column into a new 1.5ml tube. Add 50μl RNase-free water;

15. Centrifuge at full speed for 1min and get your RNA.

(Some of materials mentioned above should be found in a purchased RNeasy kit.)