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Irina's Content

There have been 9 items by Irina (Search limited from 01-June 19)


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#29815 Anybody knows a good histone methylation inhibitor?

Posted by Irina on 21 July 2009 - 05:46 AM in DNA Methylation and Epigenetics

3-Deazaneplanocin A (AKA DZNep)

Cheers,
-Carlton


Do you happen to know where I can get it? I've read about it, it seems to be the best option for non-specific inhibition, but I can't figure out how to get it. Is it synthesized just before the experiment or can one buy it somewhere? Thanks a lot. Irina



#27690 Anybody knows a good histone methylation inhibitor?

Posted by Irina on 26 June 2009 - 11:42 AM in DNA Methylation and Epigenetics

Thanks
Irina



#25963 How stable is methylated DNA?

Posted by Irina on 04 June 2009 - 05:40 AM in DNA Methylation and Epigenetics

Thanks everyone. I'll aliquot from now on, but I guess I have to believe my results, having also repeated them. I know the Phytophora infestans, a similar pathogen that infects potato, does show some DNA methylation, so it is possible that there is some in my organism too. But I guess in the region I've looked at so far there's nothing.
I'll try also the first half of the gene (perhaps elongation would be inhibited by methylation) and if not, look in another direction.
Irina



#25766 How stable is methylated DNA?

Posted by Irina on 02 June 2009 - 06:49 AM in DNA Methylation and Epigenetics

Hi everyone,

My question may be stupid, but since I'm new at this I'll ask it anyway: how stable is methylation of DNA? I have made DNA preps about 2 months ago, and since then I have been using the EZ methylation kit to try and figure out if there is any methylation in the promoter of my gene. I'm surprised to see all Cs change to Ts in my sequencing results. I've increased the amount of DNA that I treat in case it wasn't sufficient and everything got changed because of that. I'm now at 500ng per reaction, which is the high end of the optimal range recommended.
Also, how frequent is DNA methylation? Is it normal that promoters wouldn't have any methylation at all? I work on Phytophthora sojae, a pathogen of soybean, and unfortunately I can't find any gene that is known to have methylation, to use as positive control. So far I've worked up over 1000bp upstream of start ATG and see no methylation at all.

Thanks a lot
Irina



#23718 EZ DNA Methylation kit

Posted by Irina on 07 May 2009 - 11:31 AM in DNA Methylation and Epigenetics

Is there any possibility that the bisulfite treatment would "bypass" methylation, and convert even methylated cytosines to uracil? I've used the Zymo EZ DNA Methylation-Gold kit, and followed their specifications exactly. I get nice sequences, with every single C changed to T. I was just wondering if that was a possibility. I have no reason to think anything went wrong though.
Thanks
Irina



#23196 Probe size for miRNA Northern blot?

Posted by Irina on 01 May 2009 - 05:10 AM in siRNA, microRNA and RNAi

Thanks a lot. I'll try both ways.
Irina



#22975 Probe size for miRNA Northern blot?

Posted by Irina on 28 April 2009 - 08:58 AM in siRNA, microRNA and RNAi

Jon> I'll look into your suggestions and try to find some programs. I'm very new to the whole miRNA issue, and I'm apparently taking the hardest road to learn it :-)
Functional Screens> I don't even know if there is a miRNA. It's just a possiblity I'm trying to test. That's why I'm having so much trouble finding the right approach to figure out if there is one. If you have a better suggestion, I'd be more than happy to take it :-)
andrea> I'm working on a pathogen of the soybean.
Thanks for all the help.
Irina



#22858 Probe size for miRNA Northern blot?

Posted by Irina on 27 April 2009 - 05:05 AM in siRNA, microRNA and RNAi

Sorry, I don't really understand your question.
You want to determine the knockdown efficiency or you want to detect the miRNA?
You knock down gene (mRNA) by shRNA or miRNA?
Do you think your gene of interest is silenced by a unknown miRNA?



My gene of interest may be silenced by miRNA. I'd like to test that possibility. I've run a 15%PAA 7M urea gel, and the RNA looked good. I transferred it and I'd like to now do a Northern blot, but I'm still working on deciding a good size for the probes. Obviously, I have no idea where such and RNA would be "located" (with respect to the full length mRNA).
Thanks
irina



#22554 Probe size for miRNA Northern blot?

Posted by Irina on 23 April 2009 - 05:11 AM in siRNA, microRNA and RNAi

I'm tryting to determine if my gene is silenced using small RNAs. I'm planning to do a Northern blot (I've already run and transferred the gel). I have no idea if there is any miRNA, so I'm going to have to scan throughout the entire length of the gene but I don't know the size of the probes I should use. I'd really appreciate if anyone could help with that.
Thanks a lot
Irina




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