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My Northern Blot doesn't work for DNAsed samples - (Mar/29/2008 )

I have isolated total RNA from transfected mammalian cells using Trizol/Chloroform/Isopropanol method, and got plenty of RNA. I took aliquots of the samples and DNAsed them using Ambion TurboDNAfree, then standard ammonium acetate/ etoh pptn, with glycogen (Glyco-Blue) as carrier. Yields were as expected, and gel for Northern looks great, with all samples (both DNAsed and not DNAsed) intact and high quality. (All look identical on gel, with nice bright 18s and 28s bands, small RNA bands ~100-200nt, and no smears.) When probed, however, only the non-DNAsed samples give a signal for the transcript. My transcript is relatively small (220bp), and as I understand it, any DNA in the sample could not have migrated in the formaldehyde denaturing gel. (I did run some plasmid to test this, and it stayed up near the 28s band -- but I have not tried smaller DNA fragments.)

The only difference between the samples (other than DNAse treatment and subsequent pptn) is that the DNAsed samples were precipitated with glycogen. Could this be interfering with hybridization of the probe? (I'm using a DNA probe, cut from plasmid.) Or am I somehow losing my transcript in the second precipitation? (have used amm acetate/etoh before many times with no problems, but never for a transcript this small...) Or am I getting false positives in my non-DNAsed samples?
Please help...

Thank you in advance blush.gif


I just had a thought -- in the second precipitation, done with the DNAsed samples only, what would be the effect if something was wrong with my 5M ammonium acetate? (It was made in-house). How would this effect my RNA? unsure.gif


Why are you doing an ammonium acetate precipitation? You're already precipitating the RNA by doing the Trizol extraction (adding the aqueous phase to the isopropanol is the precipitation step...). You should just be able to wash your pellet with 75% EtOH and be done with it. Don't add extra steps or chemicals if you don't have to.


Thanks for your reply... I agree with you -- but I'm only doing the ammonium acetate/etoh precipitation on the samples that were DNAsed after the first precipitation. (It's required to re-precipitate after DNAse and deactivation of the enzyme.)

I'm setting up a series of experiments this week to try to identify the problem. Is there a better precipitation or DNAse treatment that I should be using? I've never had this problem, and I'm stumped... blink.gif

Would smaller DNA fragments (say, maybe a wayward plasmid insert?) migrate through a formaldehyde denaturing gel?? What if I precipitated some salts with the RNA in the second pptn? Could that effect the denaturation of my RNA?
Has anyone ever had a similar problem?