Hello Sarah

I will add to the above with one final  comment: Most DNAses apart from Ambions Turbo principle are highly purified and incorporate remnants of RNAses. Irrespective of particular issues with contaminated stop buffer therefore you often see nominal RNA degradation when you DNAse treat RNA with these purified principles. In addition, heating DNase to inactivate can in the presence of divalent cations not mopped up by stop buffer = EDTA induce hydrolysis of RNA. Incubating with the DNAse e.g Sigma Amplification grade DNase can help but doesnt eradicate problem completely. Ambion divests itself of RNAse because the DNAse ORF is slectively cloned expressed and purified leading to no DNAse contamination. Having used lots of purified principles and also the Ambion enzyme I can confidently say that Ambion is the only enzyme where I have NEVER seen degradation. I speak as somebody who has worked on large transcriptomic projects and evaluated many DNases for the reasons given

Obviously the problem is the STOP solution, most probably the EDTA solution was prepared with water without DEPC (diethylpyrocarbonate) treatment. Try to prepare your EDTA 5 mM with water treated with DEPC to solve your RNA degradation.

Regards.

Hi Sarah,

as Aaron already mentioned a contamination of the solution with RNAse would explain this result, however, the solutions provided by the companies (e.g. Sigma) are usually certified RNAse free. Other factors that could lead to RNA degradation is not properly buffered solutions or not adding enough. RNA is prone to alkaline hydrolysis which is further exacerbated by the presence of metal-ions such as Mg and temperature. Check if the pH and final concentration are right (you can post stock concentrations, final volume and ul stop solution added if you want).

An alternative to DNAse digestion is the precipitation of your RNA with LiCl which quite specifically precipitates RNA and usually yields clean RNA with only low amounts of DNA and protein contamination. If you omit the stop solution and heating step you can even do the DNAse treatment and immediately proceed with the LiCl without the inactivation step.

Add LiCl to a final concentration of 2.5M and mix well

Incubate at -20C for 30min

Centrifuge for 20min, >16,000g @4C

Wash with one volume 70% ethanol

Air dry pellet and resuspend in desired volume RNAse free water

One word of caution. LiCl precipitation has limitations in that it doesn't efficiently precipitate very small RNA's (10ng/ul RNA)

Let me know if it helped.

Manuel

Aaron: Yes, it definitely looks as if the STOP solution is contaminated; however, new STOP solution yields the same result, so that left me wondering.

Manuel: I'm storing the stock RNA in RNase-free water from Ambion (the water alone doesn't cause degredation--I've tested it at room temperature and run a gel to look). Perhaps as you suggest, my reaction is not sufficiently buffered, or the chelation with EDTA is not sufficient.

Here is the reaction mix for the DNase I. I used a 20ul reaction volume.

3.2ul RNA in RNase-free water (14ug RNA), 2ul 10x reaction buffer (200mM Tris-HCl pH 8.3, 20mM MgCl2), 2ul DNase I (1U/ul in 50% glycerol, 10mM Tris-HCl pH7.5, 10mM CaCl2, 10mM MgCl2). 12.8 ul of RNase-free water brings this to 20ul. This is incubated at room temperature for 15 minutes (my RNA survives this part just fine).

Then, I add 2ul STOP solution (50mM EDTA), so the final working concentration of EDTA is 5mM (or very close to 5mM).  After 10 minutes at 70 degrees, the RNA is gone.

Thank you for your thoughts on this,

Sarah

Dear Sarah,

The 4ng/ul concentration you're mentioning is very low for RNA to be stable over time. Why is this so low (to much diluting or low yield)? I would strongly recommend using RNAzol for your RNA isolation, instead of troubleshooting further. With RNAzol  no DNAse I treatment is required and higher RNA yields are obtained than any other method we've tested.

http://www.mrcgene.com/rnazol.htm 

All best

Sotiris

Sotiris:

My apologies, I should have written 4ug/ul.  Thank you for the reference to another method.  I have not tried the RNAzol and I will give it a close look.  best, Sarah

please remember that excellent kits are available with on-column gDNA elimination (with or without on-column DNAse digestion). anyway... I think that Manuel is right when suggesting to check pH... but most of all I think that you have "cryptic" RNAse somewhere.

Finally: genomic contamination is the MAJOR thing that you have to actually avoid for qPCR; genomic interference in quantification is worse than acceptable levels of RNA degradation... that's my opinion :)

I usually just design primers that span large introns, and then don't worry about genomic contamination. Primers are added in such excess that "chelation" by genomic contamination is irrelevant, and most transcripts are present at much higher levels than genomic copies anyway (though I've happily used this for even relatively rarely expressed transcripts too).

You could always design a 'genomic only' PCR set of primers and quantify your genomic contamination, if you're worried. 

Hi, I use the DNA-free™ Kit (Ambion) for gDNA removal from RNA samples. Great results, easy to use and no stop solution required.

Thank you so much, everybody.  You've given me some ideas to try!  I see that the Ambion DNase-free gets a nice review on Biocompare  http://www.biocompare.com/Product-Reviews/40311-DNA-free-Kit-From-Ambion/  .  Or, since I have to re-isolate all of my RNA anyway I could switch from Trizol to RNAzol  (no DNAse needed at all, apparently), and the RNAzol looks like its not super-pricey.  The Qiagen-type columns are convenient, but I've found them hard to scale up when I'm using lots of different kinds of tissues.

very grateful for the insights,  Sarah

Hello Sarah

I will add to the above with one final  comment: Most DNAses apart from Ambions Turbo principle are highly purified and incorporate remnants of RNAses. Irrespective of particular issues with contaminated stop buffer therefore you often see nominal RNA degradation when you DNAse treat RNA with these purified principles. In addition, heating DNase to inactivate can in the presence of divalent cations not mopped up by stop buffer = EDTA induce hydrolysis of RNA. Incubating with the DNAse e.g Sigma Amplification grade DNase can help but doesnt eradicate problem completely. Ambion divests itself of RNAse because the DNAse ORF is slectively cloned expressed and purified leading to no DNAse contamination. Having used lots of purified principles and also the Ambion enzyme I can confidently say that Ambion is the only enzyme where I have NEVER seen degradation. I speak as somebody who has worked on large transcriptomic projects and evaluated many DNases for the reasons given

One more thing. Because Divalent cation induced hydrolysis of RNA is well recognised phenomenon the stop principle in Ambion recombinant DNAse I is not based on heating. Thus, with Ambion rDNAse I you avoid digestion of RNA by virtue of contaminating RNases and also subsequent divalent cation hydrolysis of RNA. In short, ALWAYS use Ambion DNAse I

Thank you Laurence. I feel pretty sure that there is some hydrolysis due to the divalent cations in solution, and exacerbated by the heating.  As a non-biochemist there is only so much troubleshooting I can do capably, so I'm going to try the Ambion product.  best, Sarah

Followup:  I tried the Ambion DNase-free kit, which is advertised to remove DNA contamination from RNA without requiring a high temperature incubation to inactivate the DNase enzyme.  It got rid of genomic contamination and my RNA looks good! 

I've attached an agarose gel (it won't win any beauty contests).  Tried to load about 1ug RNA per lane. Lanes 1 and 2 are duplicate samples of the spleen RNA before DNAse treatment, with genomic contamination visible.  Lanes 3 and 4 are duplicates of the same RNA after Ambion DNAse-free treatment.  Lanes 5 is RNA treated with a previous DNase protocol involving heat, and 6 and 7 are various RNA samples I had hanging around the -80, unrelated to this experiment.  Lane 8 is 100bp DNA ladder.

Glad to be moving on!

Sarah

Good news Sarah !!

It is without doubt in my experience the best DNAse and there are fundamental resons for that

Love the picture of the dog !!

You could design the primers crossing exon.

Ping Xiang Thanks for your suggestion, I'll try it!