Gattuso:RNA extraction

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(New page: Decalcification To 500 ml cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring teh final concentration to 2.2 mM. Swirl the flask and note the clearing of teh culture. After ad...)
Current revision (12:30, 9 October 2007) (view source)
m (RNA extraction moved to Gattuso:RNA extraction: this is a lab-specific protocol, moving it into that namespace)
 
(2 intermediate revisions not shown.)
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Decalcification
Decalcification
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To 500 ml  cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring teh final concentration to 2.2 mM.
+
To 500 ml  cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring the final concentration to 2.2 mM.
-
Swirl the flask and note the clearing of teh culture. After adding the HCl, check if teh pH has dropped from 8.0 to 5.0 using pH strips.
+
Swirl the flask and note the clearing of the culture. After adding the HCl, check if the pH has dropped from 8.0 to 5.0 using pH strips.
After 1 min, quikly neutralize the solution by adding 14.3 ml of 0.1 M NaOH (final concentration of 2.8 mM). This serves to  restore the culture to a neutral pH of 8.0.
After 1 min, quikly neutralize the solution by adding 14.3 ml of 0.1 M NaOH (final concentration of 2.8 mM). This serves to  restore the culture to a neutral pH of 8.0.
Line 12: Line 12:
Resuspend the cells in 100% ethanol and pellet the cells by centrifugation before storing at -20°C.
Resuspend the cells in 100% ethanol and pellet the cells by centrifugation before storing at -20°C.
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2- if teh RNA is to be extracted immediatly
+
2- if the RNA is to be extracted immediately
Resuspend and combine the cells in a disposable centrifuge tube using centrifugation.
Resuspend and combine the cells in a disposable centrifuge tube using centrifugation.
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1- Deep the tube  in liquid nitrogen few minutes
1- Deep the tube  in liquid nitrogen few minutes
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2- Grind the cells into fine powder using a grinder adapted to the size of the tube.
+
2- Homogenize the sample using a seringe and needle (21G).
-
3- Add 500 µl of extraction buffer and vortex for 30s. vent to release any pressure built up.
+
3- Add 500 µl of extraction buffer* and vortex for 30s. Vent to release any pressure built up.
4- Add 50 µl of 2 M sodium acetate, pH 4.0 and vortex for 30 s.
4- Add 50 µl of 2 M sodium acetate, pH 4.0 and vortex for 30 s.
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8- Remove the RNA contained in the upper aqueous phase and add an equal volume of isopropanol. Mix and incubate at -20°C for at least 45 min.
8- Remove the RNA contained in the upper aqueous phase and add an equal volume of isopropanol. Mix and incubate at -20°C for at least 45 min.
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9- Pellet teh RNA by centrifugation at 10000 g for 10 min at 4°C
+
9- Pellet the RNA by centrifugation at 10000 g for 10 min at 4°C
10- Resuspend in 25 µl sterile water. Add an equal volume of 4 M LiCl and mix. Place on ice for 45 min.
10- Resuspend in 25 µl sterile water. Add an equal volume of 4 M LiCl and mix. Place on ice for 45 min.
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11_ Centrifuge for 5 min at full speed. Wash the pellet in cold 70% ethanol, air dry and rewsuspend in small volume (50 µl-100 µl) of water
+
11_ Centrifuge for 5 min at full speed. Wash the pellet in cold 70% ethanol, air dry and resuspend in small volume (50 µl-100 µl) of water
 +
 
 +
*GITC buffer (RNA extraction buffer)
 +
4M guanidium isothiocyanate
 +
25 mM sodium citrate
 +
0.5% sarkosyl
 +
0.1% beta-mercaptoethanol
 +
 
 +
The buffer should be prepare using nanodrop water and autoclave before adding the beta-mercaptoethanol.

Current revision

Decalcification

To 500 ml cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring the final concentration to 2.2 mM. Swirl the flask and note the clearing of the culture. After adding the HCl, check if the pH has dropped from 8.0 to 5.0 using pH strips. After 1 min, quikly neutralize the solution by adding 14.3 ml of 0.1 M NaOH (final concentration of 2.8 mM). This serves to restore the culture to a neutral pH of 8.0.

Haversting cells Centrifuge at 10000g for 10 min at RT. Do not place the cells on ice as this will cold shock the cells and activate specifics RNAses within the cell

1- if the RNA is to be extracted later: Resuspend the cells in 100% ethanol and pellet the cells by centrifugation before storing at -20°C.

2- if the RNA is to be extracted immediately Resuspend and combine the cells in a disposable centrifuge tube using centrifugation.


RNA extraction

1- Deep the tube in liquid nitrogen few minutes

2- Homogenize the sample using a seringe and needle (21G).

3- Add 500 µl of extraction buffer* and vortex for 30s. Vent to release any pressure built up.

4- Add 50 µl of 2 M sodium acetate, pH 4.0 and vortex for 30 s.

5- Add 500 µl of water saturated phenol (pH 4.3) and vortex 30 s.

6- Add 100 µl of chloroform:isoamyl alcohol (24:1) and vortex for 30 s.

7- Centrifuge at room temperature for 10 min at 5000 g to precipitate protein and DNA.

8- Remove the RNA contained in the upper aqueous phase and add an equal volume of isopropanol. Mix and incubate at -20°C for at least 45 min.

9- Pellet the RNA by centrifugation at 10000 g for 10 min at 4°C

10- Resuspend in 25 µl sterile water. Add an equal volume of 4 M LiCl and mix. Place on ice for 45 min.

11_ Centrifuge for 5 min at full speed. Wash the pellet in cold 70% ethanol, air dry and resuspend in small volume (50 µl-100 µl) of water

  • GITC buffer (RNA extraction buffer)

4M guanidium isothiocyanate 25 mM sodium citrate 0.5% sarkosyl 0.1% beta-mercaptoethanol

The buffer should be prepare using nanodrop water and autoclave before adding the beta-mercaptoethanol.

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