24 replies to this topic

[stardust]

Hi,

RNA looks fine after the 37°C incubation step. If there is some DNA left, i usually repeat the Turbo RNA procedure. RNA is still fine. You just have to work clean before...

Stardust

[Jack111]

marek82313, on Jun 19 2009, 08:37 AM, said:

I everybody,

I have RNA samples with genomic contamination too, and I sometimes can't rid of it. I use the Promega RQ1 but usually I proceed purifing the samples by column or reprecipitating the RNA with isopropanol, because I thought the salts contained in its buffer could be annoying for the retrotranscription. Is it so? Or am I doing something unuseful?
Is the RQ1 DNAse completely inactive after incubation at 65°C and addition of the DNase stop buffer, or could a minimal extent of activity persist?
I usually check genomic DNA contamination by performing PCR reactions with 2 or 3 primer pairs.

Thanks in advance

Marco

Hi, I have same problem like you and I tried different methods(including what you introduced). Unfortunately, it also doesn't work. Finally, I find a vedor to synthesis it. I think DNA synthesis(http://www.genscript..._synthesis.html) is very cost-effienct, becasue my DNA fragment is not long.

[William Parkar]

If you want to determine if you have dna present then treat with dnase and reprecipitate the rna to get rid of the dntps generated. then determine how much rna is present.


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[shane]

which can be got relieve of. generally when the no. of units for RNA extraction is high, gDNA inadvertently arrives up throughout extraction.

[lzheng]

I need RNA to construct a standard curve for my RT-qPCR. The RNA is produced via in-vitro transcription using a cDNA template but there's always a large amount of cDNA contamination in the cRNA. I have tried successive DNase treatments (x3 times) in an attempt to remove the DNA from my RNA transcripts. My problem is that once I have removed enough DNA from the RNA transcripts to keep it at an undetectable level, I also have very little RNA left (i.e. 3ng/ul).

I have used Lithium precipitation to recover the RNA after each DNase treatment. I seem to lose a large amount of RNA in the precipitation step. I have also used RNase columns (qiagen) to recover the RNA but with no improvement on the RNA recovery rate.

This low concentration of RNA cannot be used in the construction of a standard curve.

I do not understand how others (i.e. published papers) can produce DNA-free cRNA seemingly with ease with only one on-column DNase treatment of the RNA whereas 3 DNase-treatment was needed in my samples.

Can anyone who has produced DNA-free cRNA help me out?

Thanks.

[HomeBrew]

The first thing I would try is to skip the RNA recovery after each digestion -- just digest it three times, adding DNAse as appropriate to the volume each time, and recover the RNA once after you're done with the third digestion (or do one digestion with three times the amount of DNAse?).

If you still lose a lot of RNA with lithium precipitation, consider adding a carrier molecule like oyster shell glycogen to improve recovery, or try column recovery.

[Sagesigh]

I think our Pippin Prep instrument could, in theory, cleanly extract RNA from genomic DNA. Will check with our development group about a making special precast cassette for this purpose. If we build a cassette for this purpose, is anyone interested in beta testing?

What are the relative sizes of the RNA and DNA in question? Easy to parse electrophoretically?

[tilia25]

Hi all!

I use Qiagen RNeasy kit with on-column DNA digestion step and it removes all DNA only in 20% of the cases. This is why I follow up with TurboDNase kit with EDTA addition and heat deactivation. The DNA is removed but I encounter another problem.. After cDNA synthesis the PCR doesn't work! I checked up all possibilities (RNA degradation, cDNA synthesis fail, PCR products etc), everything leads to Turbo DNase buffer..
To test it, I used DNA template that works and mixed it with one of the cDNA samples that does not give results, also I added aliquot of DNase buffer to my DNA sample and included positive control (just the DNA). After standard PCR, only positive control gave good band; the mix between DNA and cDNA showed very faint band and third sample failed completely! Did anyone have similar problems?
I tried to purify my cDNA on a column, precipitate, add betaine, caseine, BSA, adjust Mg2+ concentration, dilute, add 1% Triton-X.. all for nothing.
Only 10x dilution and addition of 1% Triton gave me very faint band.. and in 2 cases (out of 40) precipitation restored template activity.
After column purification or precipitation my samples are clean (perfect spectra on NanoDrop) and of ca. 30 ng/ul concentration. Gels of RNA and cDNA look good too.

I am desperate.
Please help!

[WSN]

The straightest way to differentiate amount of DNA from RNA is to run RT with and with out reverse transcriptase in it, the delta Ct between them is the indication of the RNA amount, while Ct from RT free indicates the DNA amount. RNAMono assay from Metammune.com exclusively amplifies RNA from DNA background, go check their design, you can design your own assay their way.

Edited by WSN, 06 January 2012 - 09:48 AM.

[abigen]

you can use DNase I

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