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RNA extraction from plant cell cultures - (Mar/17/2008 )

Hi I am new to the board I I just look for some help about the RNA extraction from plant cell cultures. Well I never have extracted RNA from plant cell culture, but just from leaves and the trizol protocol I used was a 6ml trizol to 1g of leaves.
The trizol protocol for cells says to use 0.75 of trizol to 5 - 10 x 106 cells there are any way I know how much this cells weight? there are kind of convertion?

Or there are anyone how use a good trizol protocol to plant cells?

Thanks.

-pauloschlogl-

Hi...

I extract RNA with the following protocol

*Cut the leaves and weight them. Immediately after doing so, place them into liquid nitrogen。
*Grind the samples inside a mortar that was previously cooled with liquid nitrogen. Add extraction buffer (3 x) and phenol (3x). Work as quickly as possible at this point.
* Centrifuge at 15000 rpm for 10 min at 4 degrees
* place the supernatant into a new tube and add acid phenol and chloroform (1/2:1/2)
*Centrifuge at 15000 rpm for 10 min
*Rescue the supernatant and add it LiCl 10 M (1/3). Vortex and take it to -20 C. (from an hour to OVERNIGHT.)

*Centrifuge at 15000 rpm for 10 min
*Discard supernatant and add to the pellet 1000 microl of 2M LiCl. Vortex
*Centrifuge 15000 rpm for 15 min
*Discard supernatant and add 100 microl of TE buffer, 10 microl of sodium acetate 3M
*let it rest on ice for about 10 min
*Add phenol/chloroform (50 microl/50)
*Centrifuge at 15000 rpm for 10 min
*place the supernatant into a new tube and add 100% ethanol (2.5 x)
*let it rest on ice for 10 min (For the ARN to decant)
*Centrifuge at 15000 rpm for 15 min
*Discard supernatant and add to the pellet 70% ethanol (1000 microl)
*Centrifuge at 15000 rpm for 10 min
*Discard supernatant and dry the pellet.
*Add 20 microl of TE buffer and store at -80 C


I have done it this way for several years and had no problems at all.
Hope it helps you

-solmaniar-

You can use a counting chamber under a microscope. Here's a link on the subject: http://www.ruf.rice.edu/~bioslabs/methods/...llcounting.html

With that you can determine the amount of cells per mL.

-Ambrósio-