I have a problem to isolate high-quality RNA from the secondary needles of Norway spruce (Picea abies) for real-time qPCR. I used InviTrap Spin Plant RNA Mini Kit, it works perfectly for RNA isolation from the spruce cotyledons, but absolutely not for secondary needles, there is almost no detectable RNA content. I tried to use also classic phenol-chloroform extraction, the RNA concentration was sufficient, but it was really problematic in the next steps (cDNA synthesis and the qPCR itself was not successful. So my question is, if you have some experiences how to isolate high-quality RNA from the spruce secondary needles usable for real-time qPCR analysis? Thanks a lot!
I am using sigma trizol reagent for total RNA extraction. It gives high quality RNA. Further Check the RNA quality gel running. if the RNA is of good quality means try the cDNA preparation protocol on the same day it self. i feel the storage of RNA may lead to significant lose of RNA. does the GAPDH or Actin does amplified in PCR ? if this endogenous control also has not amplified means your RNA was completely degraded I think ! Always use fresh plant material. Do everything in a cool condition using ice. Still you see the degradation of RNA means prepare chemicals and reagents freshly. Q PCR not successfull means what the length of the amplicon does your primers amplifies ? use primers those give 80-90 bp amplicons. all the best.
Dear Dinesh, I can make you sure, that Trizol or Trisure systems absolutely don´t work for gymnosperm plants samples. There´s no, or completely degraded RNA. On the other hand using the Kit mentioned above is fine and efficient for isolation from spruce cotyledons, but it doesn´t give any RNA from the secondary needles. In the case of cotyledons qPCR was successfull, reference, as well as "my" genes very expressed as I´ve expected, there was no problem. For both I can say that I work carefully, in completely RNase-free conditions, on ice... So I can´t accept the opinion it could be caused by incorrect handling :)
For Joke: I lyse my cells (tissue) using Retsch mill, pre-cooled; also my samples are taken straightly from liquid nitrogen. So it makes me sure that the RNA-isolation problem is not caused by insufficient disruption of cells, but probably there´s some biochemical problem, why the techniques I used are not suitable for secondary needles samples. I tired to use also classic phenol-chloroform extraction (acidic phenol) and again, it works fine for the spruce cotyledons but not for needles, I think because the very high amount of some secondary metabolites.
Anyway, thank you both! ;)
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