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Protocols: ES Cell Culture - Gene Targeting - Protocols

Preparation of RNA from Cultured Cells

Notes:	When preparing solutions for use with RNA, use only 
MilliQ water and disposable sterile Tissue Culture plasticware.  
Also, if preparing working solutions from stock solutions 
(e.g., 0.5 M EDTA pH 8.0 or 1.0 M NaCl), make sure that the stocks 
were also prepared in sterile plasticware with MilliQ.

		To rinse the homogenizer, set up a series of 3 Erlenmeyer 
flasks.  Fill each with MilliQ water and add a little SDS to the first 
flask.  To rinse, go from the first flask to the next, turning the 
homogenizer on at each step.

	1.	Wash the cells twice with PBS.  Add 3 ml trypsin per 10 cm 
plate, swirl to cover the surface, and incubate for 10 minutes.

	2.	Add 3 ml of M15 to neutralize the trypsin and resuspend the 
cells by vigorous pipetting.

	3.	Pool the cells in a 50 ml polypropylene tube.  Pellet the 
cells by centrifugation @ 1,000 rpm for 5 minutes.

	4.	Aspirate off the supernatant and add 8 ml of ice-cold 
Lysis Buffer (6 M Urea, 3 M LiCl, 1 mM EDTA pH 8.0 in MilliQ water).  
Keep samples on ice.

	5.	Homogenize samples on ice @ maximum speed for 1.5 minutes 
to destroy the cells and break down the genomic DNA.  Rinse the 
homogenizer between one sample and the next.

	6.	Transfer the lysates to 15 ml polypropylene tubes 
(these tubes must be able to resist high-speed centrifugation).  Place 
the tubes in an ice bucket (with ice) and store overnight @ 4o C.

	7.	Centrifuge the samples @ 10,000 rpm for 30 minutes @ 4o C 
(use the Sorvall HB-4 or SA-600 rotors; adapters will be required for 
the tubes).

	8.	Discard the supernatant.  Invert the tubes onto paper towels 
to thoroughly drain.

	9.	Resuspend the pellet in a total volume of 0.4 ml  of  
Solution (10 mM Tris pH 8.0, 0.5% SDS prepared in MilliQ water).  
Add the solution 0.1 ml at a time, resuspend the pellet, and transfer 
the RNA to microcentrifuge tubes on ice.  Repeat with subsequent 0.1 ml 
volumes, transferring each wash to the microcentrifuge tubes, until the 
total 0.4 ml has been added.

	10.	Extract  once with an equal volume of phenol, once with an 
equal volume of phenol/chloroform, and once with an equal volume of 
chloroform.  Avoid carrying the interphase between one extraction and 
the next.

	11.	Precipitate the RNA by adding NaCl to 0.15 M and 2.5 
volumes of cold EtOH.  Invert the tubes a few times and store @ -70o C 
for 1 hour (or overnight @ -20o C).

	12.	Spin samples in the microcentrifuge @ maximum speed for 
10 minutes in the cold room.

	13.	Aspirate off the supernatant and rinse the pellet with 
70% EtOH.  Allow to air dry a little.

	14.	Resuspend the RNA pellet in 0.1 ml of DEPC-treated H2O 
(DEPC=diethylpyrocarbonate; add 1 ml to 1 L of MilliQ water, mix, and 
leave overnight @ 37o C, then autoclave).  Vortex vigorously to force 

	15.	Transfer 1 ul of sample to a clean microcentrifuge tube, 
dilute 1:500 with dH2O, and read A260.  RNA concentration 
(ug/ul) = A260 multiplied by 20.

	16.	Store the RNA samples @ -20o C.  The RNA is ready 
for electrophoresis.