Chewbby

 Total RNA Extraction from Yeast

Preparation :

Cold RNAse-free water

Cold RNAse-free 70% Ethanol at -20°C

Dry bath
Phenol

Chlorophorm
Liquid nitrogen (### Wear eye protection and gloves ###)

TES Lysis Buffer:

10 mM Tris-HCl (pH7.4)
10 mM EDTA
0.5 % SDS

TE 7.5:

10 mM Tris-HCl (pH7.4)
1 mM EDTA (pH8)

IMPORTANT: 

Work under FUME HOOD with eye protection and gloves when handling phenol or chloroform.
Work in RNA dedicated area for the other steps.
Use RNAse-free pipets and tips all the way through.

Extraction Day 1:

1- Harvest cell culture at 10e7 cells/ml.

2- For small cultures : In a 2 ml tube, spin 13,000 rpm for 1min and discard supernatant.

For large cultures : Spin 4,000g for 5 min and discard supernatant then add 1ml of water, resuspend the cells and transfer them to a fresh 2 ml tube. Spin 13,000 rpm for 1min and discard supernatant.

3- Snap-freeze in liquid nitrogen and keep at -80°C over night.

Extraction Day 2:

1- Set centrifuge at 4 ° C and dry bath at 65°C.

2- Melt tube into ice and resuspend the pellet with 1 mL of cold RNAse-free water.

3- Transfer the solution into a new 1.5 ml “safe lock RNA” tube.

4- Spin at 13,000 rpm 30 sec and keep the tube on ice.

5- Discard supernatant and resuspend without bubbles the pellet with 500 μl of TES lysis buffer.

6- Add 500 μl of cooled phenol (4°C). Vortex 10 sec.

7- Incubate at 65°C for 1 hour, vortex every 10 minutes during this time and keep the centrifuge at 4 ° C.

8- Put the tube 2 minutes on ice.

9- Spin at 13,000 rpm, 10 min at 4°C.

10- Transfer the upper aqueous phase (~450 μl) to a fresh tube.

11- Add 100 μl of TES + 500 μl of cooled phenol to the solution and vortex 10 sec.

12- Spin at 13,000 rpm, 10 min at 4°C.

13- Transfer the upper aqueous phase (~450 μl) to a fresh tube. Be careful: Take nothing of the phenolic interphase.

14- Add 500 μl of chlorophorm and vortex 10 sec.

15- Spin at 13,000 rpm, 10 min at 4°C.

16- Transfer the upper aqueous phase (~400 μl) to a fresh tube. Be careful: Take nothing of the phenolic interphase.

 

17- Add 30 μl of NaCl 2M + 750 μl of cold EtOH 100% (-20°C) and vortex 10 sec.

18- Put the tube(s) at -20°C for 1 hour (or over night).

19- Let centrifuge at 4 ° C, prepare cold RNAse-free water and EtOH (-20°C) and dry bath at 32°C

20- Spin 13,000 rpm for 30 min at 4°C and discard carefully the supernatant.

21- Add carefully 1 ml of cold EtOH 70 %. DO NOT VORTEX

22- Spin 13,000 rpm for 5 min at 4°C and discard carefully all the supernatant.

23- Let dry the pellet at 32 °C for 5-10 min.

24- Resuspend the pellet into 100 μl of TE 7.5 and dissolve the pellet by placing the tubes at 50°C for 30-60 min (depending on the size of the pellet). Vortex every 7-10 minutes. Be careful: the solid pellet becomes transparent. Check it dissolves by pipetting.

25- At this point, the procedure depending of the next step : -qPCR/ -RNA seq etc...

  • For qPCR : possibility to run the sample through an RNeasy mini-column from Qiagen (see Rneasy manual). Elute in 50 μl  RNAse-free water.

  • For RNAseq (before preparation of libraries) : possibility to do an Oligo(dT) or  Ribozero procedure directly wihtout purification on column.

26- Store the tube at -80 °C.