RNA extraction from plant tissue - all method are welcome (Jun/18/2007 )

Hi all,

I have some problem for RNA extraction from flower tissue such as petal, pistil ovary...I can't use the commercial kit because the RNA yield is too low for expression study.
The most frequent problem is the degradation of RNA. I am sure that all reagents I used don't contain RNase, I clean with RNaseZap all lab, all glass and electrode too.
Well the first protocol that I used was the Hot Borate (Wan e Wilkins, 1994) and this protocol doesn't works in Hibiscus flower. So I tried TRIzol; this protocol worked only in petal but no in pistil and ovary. I think that is a problem of tissue, but I am not sure. Probably are tissue rich in polisaccharides, lipid and this component interfer with the RNA purity. It is possible?
Anyone knows a different protocol?
thanks

alice

hi...

here i send you the protocol I am using.... I extract RNA from complete flowers, siliques, and leaves. This one works pretty well and it is cost convinient. My samples are kind of big when compared with what you use. but I think you can adjust it for your experiments.

ARN EXTRACTION



*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. overnight is better)

*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


good luck

can i apply this protocol to extract RNA from fungal mycelia???

hi...

here i send you the protocol I am using.... I extract RNA from complete flowers, siliques, and leaves. This one works pretty well and it is cost convinient. My samples are kind of big when compared with what you use. but I think you can adjust it for your experiments.

ARN EXTRACTION



*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. overnight is better)

*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


good luck

hi...
sorry for the delay

the extraction buffer composition is

100mM tris-HCl pH 8
10 mM EDTA pH 8
100mM LiCl
1% SDS


Answering to T. reesei... I haven't try it on mycelia...I think it might work, it is worth trying....

good luck

thanks solmaniar

i will try it and i really surprized to see your protocol, it seems very simple for me, not using any special solutions or etc etc..definitely i will try sometimes

It really works for me....maybe , if you used a kit or buy the solutions, you could get more and cleaner RNA. But I prefer this protocol. I got it from a friend too, so the credits are for him.

good luck for both of you.