Hi all,
For an expression study I am trying to optimize RNA isolation and cDNA sythesis protokols. Until now I have tried Thermo Plant RNA Isolation Kit, Trizol method and CTAB method (Jaakola ét al.2001). Results with kit was insufficient. Trizol and Ctab methods provided good yield of total RNA but not sure about the quality. Results are below. With which one should I go on for cDNA synthesis?
Trizol nanodrop:
1100ng/ul - 260/280:2,03 - 260/230: 1.70
CTAB nanodrop:
400ng/ul - 260/280 2.06 - 260/230: 2.40
And any advise for putative contaminants?
Thanks to all.
Hi,
You can look through here previously discussion
https://www.researchgate.net/post/What_is_the_effect_of_a_low_260_230_ratio_on_the_purity_of_DNA
May be it will be helpful for you
Steemed Deniz,
your 280/260 ratio indicates the purity of the RNA relative to proteins and both methods are prety good, since the the values lie above 1.8. The 260/230 ratio indicates contamination with organic molecules (phenols and so on). The value of the Trizol protocol in in the range of what I have usually used with animal cells to perform RT-PCR with no problems whatsoever. It should yield a good enough material and as a day to day method it would be my choice. However, your 260/230 ratio with the CTAB method is better, so you may want to consider it your choice, specially since plants accumulate more polyphenols and organic compounds.
One more
http://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf
Hi,
take into account that your results with nanodrop may be good but the quality of your RNA may be bad. I would recommed to check RNA integrity prior to cDNA synthesis.
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