5 replies to this topic

[kazemi.masoume]

Hi,
I hope someone can help me.
I'm doing total RNA isolation from brain dead҆s whole blood sample using QIAgen RNA Blood Mini kit. My problem is about agarose gel electrophoresis(2%) of isolated RNA;

• I see an extra band above 18s and 28s rRNAs which is similar in all of my samples.
  
• After DNase treatment of RNA prior to cDNA synthesis, I can not see any rRNA band in agarose gel electrophoresis! Is it OK?

Thanks in advance

[AquaPlasmid]

Could you attach a gel photo? Without looking at your gel, I am pretty sure that the band above the 28s was the gDNA band. If you saw nothing after DNase I digestion, you must have endogenous RNase contamination of the RNA prep. Try incubating an aliquot of the RNA prep with only the DNase I buffer containing 0.1 mM Ca2+ (don't add DNase I) ,and then run the gel. I bet you will see that the 28s and 18s are gone and the DNA band remains. Otherwise, your DNase I may be contaminated with RNases.

RNA extraction from whole blood is tough, especially frozen whole blood. Check out our AquaPreserve for blood RNA extraction, it is the only reagent that can extract total RNA from frozen whole blood or buffy coat collected in common anticoagulants.

[kazemi.masoume]

Thanks for your reply.
This is a photo of gel. One point that I forgot to mention is that when I perform RT-PCR on synthetized cDNA from DNase I treated RNA, it works. My primers are designed for exon-exon junction.
regards

Attached Thumbnails

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[AquaPlasmid]

Yeah, that's the gDNA on top. Even though you couldn't see the RNA in the gel (you need about 50 ng of RNA to see it) after the DNase I digestion, cDNA synthesis and RT-PCR can work with a few ng of RNA. But it could be inconsistent due to trace RNase contamination. Did you get to try incubating the RNA prep with DNase buffer alone to see if you lose the RNA? It'll tell you if there is RNase contamination. Best of luck.

[kazemi.masoume]

Hello AquaPlasmid.
I incubated a RNA prep with DNase buffer alone. After loading on gel I saw the 18s and 28s rRNAs. To become sure about my Dnase contamination, I also treated a control RNA (from the cDNA synthesis kit that I previously checked it on gel) with Dnase, this time there was no RNA band again. Do you think that my DNase is contaminated with RNase and is it possible to use RNase inhibitor during DNase treatment?

[AquaPlasmid]

Yes, it sounds like your DNase I is contaminated. Maybe you need to order a fresh one from another vendor. Ambion's Turbo DNase and Qiagen's are all very good. How much DNase I did you use in a 10-20 ul reaction? You don't need a lot DNase I, just pipet in ~0.5 ul DNase I and then expel it all out, then pipet a couple times to rinse your tip in the reaction mix is enough. Too much DNase I could also chop up the RNA.