Tuesday, April 14, 2009

how to extract RNA from biological samples


This is a protocol I'm currently using which was developed by two PhD student friends and a postdoc. Its really detailed so I thought I'd share it with the world because a lot of this stuff isn't intuitive and is only understood after much trial & error.

By the way, this protocol doesn't work for plant material. Planty RNA is protected by thick cell walls and is not easily isolated from the rest of the planty rubbish you find in there.

RNA Extraction with TRI reagent

1. Homogenize tissue in 0.5ml of TRI (or put on ice to defrost if this has been done)
- Stand for 5 min
- Centrifuge for 5 min @ >10,000 @ 4degC
- Remove supernatant if messy sample and place in a new tube. Dispose of pellet
- Don’t use polytron homogenizer if extracting DNA – shear forces can break strands

2. Add 0.1ml Chloroform
- Shake for 15 sec (vigorously).
- Stand for 15 min (room temperature)

3. Centrifuge 15 min @ 12,000 @ 4degC
- Extract RNA phase (top/clear) into fresh tube straight away!
- Avoid white cloud above white layer as this is genomic DNA.
- If has been standing to long, or there are still bits in supernatant, then
- spin down again.

4. Add 0.25 ml Isopropanol + 1 ul LPA (linear polyacrylamide – coprecipitant for very small amounts)
- Stand for 10 min @ room temp OR over night @ -20degC
- Centrifuge 30 min @ 13,000 @ 4degC
- Mark where pellet is with marker
- Pour Isopropanol gently out into a beaker, keep an eye on pellet. Don’t lose it!

5. Spin 30 min @ >13000 @ 4degC
- Decant supernatant

6. Wash “Back down” with 1 ml 70% Ethanol
- Vortex
- Centrifuge 10 min @ 13,000 @ 4degC
- Decant Ethanol – pour off gently onto blue roll, watch pellet.
- Make sure pellet re-attaches

7. Repeat Ethanol wash
- Allow pellet to dry - upside-down on blueroll (not in sun)
- Try to take as much of EtOH as possible so doesn’t take too long to dry
- Dry completely – approx. 1 hour. But not too long.

8. Resuspend RNA with H2O (in minimum amount of water)
- 8-40 ul dependant on pellet size
- If pellet is hard to dissolve, heat solution for 10 min at 55-60 degC, then aliquot
- immediately and freeze at -80.

9. Transfer to clean 0.5ml tube and freeze at -80
- Don’t transfer if low amounts.
- Aliquot solution immediately and freeze at -80 – RNA is very unstable at room temperature.

After this step you have to remove DNAases and then create cDNA from the RNA.

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