Genomic DNA contamination of RNA - (Jun/10/2009 )

This may seem like an odd question, but is there any way of figuring out the amount of contaminating DNA in my RNA samples?

I was considering using the Nanodrop spec. and measuring using the DNA setting instead of the RNA setting, but as these are RNA samples I wondered how accurate my reading would be. Can the spec. differentiate between the two types of nucleic acid well enough to give me an accurate reading?

My ultimate aim is to have a reading of DNA content in my RNA samples before and after DNase treatment, just to put my mind at ease.

Thanks in advance for any help!

the spec can't differentiate between dna and rna. the different settings are for the calculations that are performed to quantify the sample.

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.

You could also try Invitrogen's Quant-It Pico Green dsDNA quantification kit, if you have a fluorescence plate reader.

I use promega RQ1 DNase to treat my RNA samples and incubate it at 37 overnight, but I still find gDNA contamination in a lot of my samples, so I cannot proceed to synthesize cDNA...help!!

umam on Jun 13 2009, 08:16 AM said:

i use the invitrogen DNAse and incubate it for 10 min. before purifying it on column. though, it turns out be quite efficient in some cases, in some others gDNA contam. still persists. i guess, there is an extent of contam. which can be got rid of. usually when the no. of cells for RNA extraction is high, gDNA inadvertently comes up during extraction. therefore, in such cases i split up my RNA sample and treat each with the DNAse (basically using double the amount of DNAse than prescribed in the protocol). this might help.

Which DNAse is the best in your opinion?
I am thinking about Promega RQ1 because re-precipitation is not required after its treatment.
Umam, hows promega DNAse? Do you follow the standard protocol recommended by the company or change it a bit? How much RNA you add for cDNA synthesis after DNAse treatment?

Hi,

I use the Turbo DNA free kit von ambion. Works well and is easy. Basically you add DNAse I and Buffer, incubate for 30 min at 37°C then you add their bead based inactivation reagent, incubate, spin and take the supernatant. For me it work 80 - 90% of the time.

Stardust

Ok I'll try thr turbo DNase.

Signal : For my promega RQ1 I use upto 1 microgram RNA before treating it with Dnase and the kit asks for 30 min incubation with RQ1, but I incubate it overnight at 37C and I also add Rnase inhibitor that does not come with the kit.
-Uma

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

marek82313 on Jun 19 2009, 09:37 AM said:

DNase digestion should be fine. Heat inactivation should be fine. DNase enzymes are not the most robust enzymes and can denature by simply vortexing or freeze/thaw...if you're too aggressive with the enzyme maybe that is why you cannot get rid of all of the gDNA contaminating your sample???

You don't need to add that extra step after inactivation...switch to on-column DNase digest if you want to worry less about salts and DNase.

Just do the usual control PCR reactions...you only need one primer pair if they span an intron...if this isn't an option and you can only amplify from within an exon, do a NO RT control...if you get amplification in the NO RT, then you have contaminating gDNA.

Use a bioanalyser if you have access to one if you want to see the gDNA contam in your sample.