RNA extraction problem

[MedGen]

Okay, I'm extracting RNA from whole blood samples. I've done a red cell lysis and got my RNA extracted via a modified version of the Chomsinsky method. The problem is that I'm co-extracting far too much DNA with the RNA.

 

I ran the samples down a gel, and I've got the 28S and 16S rRNA bands as well as a lot of genomic DNA contamination. I've run a second extraction against phenol:chloroform. I've just added the isopropanol and there's an aggregate forming instantly, which to me screams of DNA contamination. I've not DNase treated the samples yet because there seemed to much contamination to get rid of it all sufficiently.

 

I need to get this extraction really quite homogenous in terms of RNA: DNA because of the downstream procedures I'm running.

 

I've nanodropped the samples and they all get OD260/280 of 1.89-1.90.

 

Is there any modification I can make that will reduce this DNA contamination significantly?

 

Thanks in advance.

Edited March 11, 2009 by MedGen

[DrDNA]

First of all, ALL RNA isolation methods yield residual DNA.

Popular methods include DNase I digestion, phenol:chloroform extraction, or lithium chloride precipitation. All of these have been shown to be successful are removing DNA from RNA.

I believe that DNase I digestion and/or DNase I digestion followed by one of the other methods often works the best.

[ecoli]

Just corroborating DrDNA. You need to do DNase digest before doing anything useful with RNA.

[CharonY]

Chomczynski is the acid phenol-chloroform method. Standard phenol-chloroform yields DNA as well as RNA. In fact, this is something that may have happened. One thing to check is whether the pH is right. If it is basic, then the DNA will be coextracted in very high concentrations. And are you sure not to have any protein contaminations?

 

Otherwise it is possible that there is indeed an extremely high DNA to RNA ratio. An easy cleanup would be DNAse digest on silica spin columns. Though each cleanup will result in loss, obviously.

 

But the first thing to do is to check the reagents.

 

Edit: cross posted, but just wanted to add that depending on sample, protocol and intended use not always DNaseI digest is required. But obviously one needs to check it via PCR to be sure.

[MedGen]

I'm not talking about some residual DNA, I'm talking about a visible aggregate as soon as the isopropanol is added, and I mean immediately visible, even after a second phenol extraction.

 

We checked all the reagents. Acid phenol was at pH4.5, all the other reagents we made up fresh today, there just appears to be a crap load of DNA in these samples. I'll DNase them and see how they come out afterwards.

 

We've had the idea of physically removing the visible DNA aggregate from the samples (yes it is that big) by pipette. I know this may result in some RNA loss, but it's worth it for the purity of the isolate.

[CharonY]

We've had the idea of physically removing the visible DNA aggregate from the samples (yes it is that big) by pipette.

 

Hm my first guess was that the reagents were bad and you co-isolated DNA with the RNA.

But alcohol precipitation should precipitate RNA as well as DNA. Even if the majority of the precipitate is DNA you will remove all the RNA together with the pellet.

Did you already isolate from the same sample volume RNA earlier and got less nucleic acids in your precipitate? It is probably too late already for quantitative analyses, especially if you are using an amplification method downstream (e.g. QPCR or for microarrays). You may want to check what you really got in your pellet, i.e. ensure that it is really DNA or whether there may be cross-contamination by proteins or anything similar.

 

Edit: scratch the protein bit. I somehow missed your 260/280 ratio. 1.9 is actually a good indicator for RNA, especially if using a nanodrop (and if the solution is somwhat acidic). As you are aware the "rule of thumb ratio" for RNA is 2.0 and DNA 1.8. Depending on calibration and acidity of the buffer (as well as ionic strength) I do have found that in the nanodrop the ratio to be closer to 1.9 for RNA. It is hard to evaluate that, though as the ratios are relatively close. But if you had loads of DNA I would actually have expected lower ratio.

Edited March 12, 2009 by CharonY

[MedGen]

Thanks for the help. As I said we've changed the reagents now, still got that large clump of DNA though.

 

I'm not going to remove the actual pellet, just the massive piece of DNA floating around. I appreciate that it will remove some of the RNA as well, however, the concentration is very high already, so removing a little shouldn't be an issue.