DNA contamination after RNA extraction, which kit works best? - (Nov/25/2009 )
Hey,
I`m trying to extract RNA from lung tissue of sheep using a Promega SV Total RNA extraction kit which
includes a DNase treatment.
Afterwards we checked our RNA for DNA contamination by running a GAPDH PCR and
yes, there was a whole lot of DNA in our RNA samples left.
Does that mean that this kit is not really efficient enough or is that
standerd with commercial kits to do an extra DNAse treatment afterwards?
If not, which kit generates good RNA without DNA contamination?
Is there also a superiour DNase treatment? We are going to use again Promega stuff for
this extra DNase treatment.
Thanks
hey blackeyed, are you using a column based DNase? If yes, I'd recommend using the TurboDNase from Ambion. It doesnt involve a column and hence you save some losses from the column..It comes with a inactivation reagent and you just have to spin the RNA and take the supernatant which is ready to go for downstream processes or more of DNase digestion if needed
It`s not column based no. I`ve read about the new Ambion DNase.
So if our promega kit doesnt work, we are going to use that one.
Ambion DNase is a product which I would recommend for its simple protocol and ease of use! I've never used the Promega though !
Blackeyed on Nov 25 2009, 03:07 AM said:
So if our promega kit doesnt work, we are going to use that one.
I am about to start RNA isolation from yeast cells in blood (mice blood) . I could not find a satisfactory protocol for this purpose. Can someone suggest a protocol to start with? My yeast is Candida albicans.
thanks in advance.
Blackeyed on Nov 25 2009, 05:25 PM said:
I`m trying to extract RNA from lung tissue of sheep using a Promega SV Total RNA extraction kit which
includes a DNase treatment.
Afterwards we checked our RNA for DNA contamination by running a GAPDH PCR and
yes, there was a whole lot of DNA in our RNA samples left.
Does that mean that this kit is not really efficient enough or is that
standerd with commercial kits to do an extra DNAse treatment afterwards?
If not, which kit generates good RNA without DNA contamination?
Is there also a superiour DNase treatment? We are going to use again Promega stuff for
this extra DNase treatment.
Thanks
Hi Blackeyed,
Since you still have your Promega in your hand, try to give it another shot.
I tried promega SV Total RNA extraction kit before (for my bacteria work). The first time I tried it doesn't work well as there are genomic contamination like you. However for my second try, I use lesser amount of material input, and it does solves the genomic DNA contamination problem.
The reason I can think of is because of the lysis buffer is not strong enough to lyse all the cells and left the cells on the column, and when you doing other process, the remaining cells break slowly and gradually release the DNA content, and even after you put the DNase, the cells breaking process still going on. Thus, when you do the elution of the RNA, there is contamination of DNA.
Try reduce your sample input (or increase your lysis buffer) and see if the genomic contamination had been reduce.
Keep us update.
Adrian.
adrian kohsf on Jan 19 2010, 01:47 PM said:
Blackeyed on Nov 25 2009, 05:25 PM said:
I`m trying to extract RNA from lung tissue of sheep using a Promega SV Total RNA extraction kit which
includes a DNase treatment.
Afterwards we checked our RNA for DNA contamination by running a GAPDH PCR and
yes, there was a whole lot of DNA in our RNA samples left.
Does that mean that this kit is not really efficient enough or is that
standerd with commercial kits to do an extra DNAse treatment afterwards?
If not, which kit generates good RNA without DNA contamination?
Is there also a superiour DNase treatment? We are going to use again Promega stuff for
this extra DNase treatment.
Thanks
Hi Blackeyed,
Since you still have your Promega in your hand, try to give it another shot.
I tried promega SV Total RNA extraction kit before (for my bacteria work). The first time I tried it doesn't work well as there are genomic contamination like you. However for my second try, I use lesser amount of material input, and it does solves the genomic DNA contamination problem.
The reason I can think of is because of the lysis buffer is not strong enough to lyse all the cells and left the cells on the column, and when you doing other process, the remaining cells break slowly and gradually release the DNA content, and even after you put the DNase, the cells breaking process still going on. Thus, when you do the elution of the RNA, there is contamination of DNA.
Try reduce your sample input (or increase your lysis buffer) and see if the genomic contamination had been reduce.
Keep us update.
Adrian.
Hi Blackeyed,
Totally agree with Adrian. I am having the same issue too when I am using Epicentre MasterPure RNA purification kit (my favorite), though it is a non column based kit.
In my experience, the cell concentration is pretty critical to avoid having genomic DNA contamination in you RNA sample. And I manage to get rid of genomic DNA via two ways, which is reduce the starting cell concentration, and/ or increase the final concentration of DNAase used, particularly when the kit has been used for sometime.
Besides PCR, there is another way to check for genomic DNA contamination in your RNA sample, which is using Agilent Bioanalyzer, which is much more rapid than PCR.