24 replies to this topic

[bp_serene]

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!

[mdfenko]

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.

[gfischer]

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

[umam]

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!!

[Vini]

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

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!!

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.

[Signal]

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?

[stardust]

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

[umam]

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

[marek82313]

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

[eldon]

marek82313, on Jun 19 2009, 09:37 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

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.

Edited by eldon, 19 June 2009 - 10:40 AM.

[cellcounter]

bp_serene, on Jun 10 2009, 12:44 AM, said:

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!

I use Turbo DNase kit for getting rid of the genomic contamination.

But I guess if you are still interested in finding out DNA content before and after the DNase treatment, the way to go could be..

1. Treat with RNase to completely degrade the RNA, phenol chloroform etoh to reprecipitate any contaminating DNA and measure. (Before)
2. Treat with DNase and RNase, phenol chloroform etoh to reprecipitate any remaining DNA and measure. (After)

In this, using nanodrop would help in finding out precise amount of small DNA contamination (barring the amount that you lose during phenol/chloroform).

[BGS]

stardust, on Jun 14 2009, 12:34 PM, said:

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

Hi!
I have used Turbo DNA free kit (Ambion) as well. In our samples Turbo DNA free kit  degraded about half  of the amount of the DNA contamination in the samples. There was still some DNA left in all my RNA samples after DNA degradation

Cheers, BGS

[BGS]

bp_serene, on Jun 10 2009, 10:44 AM, said:

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!

Hi!
In case you are still interested in how to figure out the amount of contaminating DNA in your RNA samples...
For this purpose we have used the Invitrogen's fluorometer Qubit www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qubit.html
The machine is much cheaper than the Bioanalyser and you can measure RNA and DNA concentration in your samples with two different dyes.
Cheers,
Barbara

[fishdoc]

BGS, on Jul 31 2009, 09:19 AM, said:

stardust, on Jun 14 2009, 12:34 PM, said:

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

Hi!
I have used Turbo DNA free kit (Ambion) as well. In our samples Turbo DNA free kit  degraded about half  of the amount of the DNA contamination in the samples. There was still some DNA left in all my RNA samples after DNA degradation

Cheers, BGS

I've had the same issue. Used DNaseI from Sigma originally, then tried Turbo from Ambion. Turbo worked much much better than natural DNase I, but still left detectable levels of DNA. Then I read a paper somewhere that the use of the RNeasy on-column digestion coupled with Turbo got nearly all bacterial DNA out, but that a new product from Epicentre would do the same, but without having to do the on-column. It's called Baseline Zero. We've been using that since, but of course it still leaves some DNA contamination.

I've come to the conclusion purely through assumption that there is a certain concentration of DNA at which the DNase is no longer able to digest. There's still a little DNA, but too little for digestion... at least at a reasonable rate.

So I ended up digesting a high concentration of RNA (and therefore fairly high DNA concentration) with Baseline Zero, then diluted the DNase-treated RNA down. I figured the digestion would drop the DNA level down as far as it would go, then the dilution would drop it even further.

I think I would digest 2 ug of total RNA in a 20 ul reaction (so 100 ng/ul concentration of RNA). Then I would dilute the RNA down to 10 ng/ul for use in first strand synthesis. For the first strand synthesis reaction (20 ul) I would use 1 ul of the 10 ng/ul RNA. So I was diluting the contaminant DNA (after DNase treatment) 10x after DNase treatment, then 20x going into the RT reaction for a total of 200x dilution of the "lowest" DNA level possible by DNase treatment.

At each step, I ran a PCR to determine if detectable DNA was present. Without DNase treatment, there was a very strong product. After DNase treatment, there was a weaker, but still strong product. After the 10x dilution, there was a very faint product, and in an RT-negative first strand synthesis reaction, there was NO product.

Also, I'd like to add, this is all done on bacterial RNA, so I don't have the luxury of spanning an intron, so DNA elimination is very important. Everything I've read regarding DNA in RNA samples is that there is virtually NO WAY to get rid of it, you just need to get it down to a manageable level, i.e., a concentration at which it is not detectable.

[Arqwen]

stardust, on Jun 14 2009, 08:34 PM, said:

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

How is your RNA after that long at 37 degrees? I would be scared of it degrading!! I work as quickly as possible before I freeze it again!
Personally I prefer Invitrogens DNas1 I kit - I have never had any problems. Also, I think (I could be wrong here!) tht using a kit (I recommend QIAGEN RNeasy) is a bit better than TRIzol for gDNA contam - also you can get an on-column protocol with it, you could use both the on coloumn and an extra DNase treatment afterwards.