This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “RNA Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but they do not represent endorsements by, or the opinions of, BioTechniques.Molecular Biology Techniques Q&A Can I extract plasma RNA from blood collected in PAXgene tubes? (Thread 21826)
Q I want to extract RNA from the plasma of blood collected in PAXgene tubes, but the extraction kit is for intracellular RNA from whole blood. Does anyone know if I can spin the tubes and use TRIzol to extract RNA from the supernatant?
The supernatant will contain plasma diluted in the RNA preservative solution found in PAXgene tubes. Will the RNA preservative hinder the TRIzol extraction?
A It should work, but since you have a liquid sample, you may need Tri-Reagent-LS instead of regular TRIzol.
Q I added 1125 µL of TRIzol LS to 525 µL of PAXgene supernatant. The yield was ~1.5 g nucleic acid, but the RNA was not clean. The 260/280 ratio was 1.7 and the 260/230 ratio was 0.3. There was a large peak at 230 which might be guanidine or phenol. Also, the peak is not at 260 nm but is closer to 270. Why is this?
A Phenol has an absorption at A270. Maybe you should try pelleting or concentrating the cells in the RNA preservative before extraction. Does the preservative protocol say anything about this? If not, I think the RNAlater protocol discusses it.
Q I'm trying to get the “cell-free” RNA. The supernatant I'm using is the result of a primary spin of whole blood collected in PAXgene RNA tubes. I will also extract RNA from the cell pellet, but I want to see if there is extractable RNA left in the supernatant.
How can I clean up the phenol contamination and whatever is causing the low 260/230 ratio?
A To clean the RNA, just precipitate it again. TRIzol-extracted RNA often has salts or phenol contamination if the aqueous layer isn't removed very carefully.
A Repeated ethanol precipitation will not remove phenol. You need to extract the RNA solution with chloroform prior to ethanol precipitation. Most people use Qiagen RNeasy spin columns to clean RNA isolated with TRIzol. The columns will remove both salt and phenol.
Q Did you have any luck getting better RNA? I have the exact problem that you have. I used 0.25 mL of the PAXgene Blood solution with TRIzol LS to get total RNA from the blood samples, I ended up with a strange white pellet that didn't have the properties of a nucleic acid. Any advice you have would be great.
A Try pelleting the material in the PAXgene/blood mix first. The pellet is usually washed to remove the preservative, but you can try extracting the pellet with TRIzol without washing it. If the extraction still doesn't work, try washing the pellet with some RNase-free buffer, re-pelleting, and extracting that. You won't get much RNA from 0.25 mL.
Q I am interested in obtaining total RNA (including microRNAs) from the whole blood, so I don't think that pelleting the cells at the beginning is an option for me. I have been combining the extractions from multiple 0.25-mL samples. I think my main problem is coming from the salts in the PAXgene tubes when I precipitate the RNA with isopro-panol. I might try putting the aqueous layer straight onto a spin column instead of precipitating the RNA first.
A To purify small RNA on a spin filter column, you just need to increase the amount of ethanol used in binding.
The very first step of the PAXgene protocol is to pellet the cells and debris. I would speculate that the total RNA is not free yet. If it were, the protocol wouldn't work. So the miRNA and total RNA are still safe inside the cells and pelleting the sample should not be a problem.
The next step after pelleting and washing the pellet is to lyse the cells in a mixture of proteinase K and chaotropic salts—that is when the RNA and DNA are released. In your case, you can lyse the pellet with TRIzol.
A We do a lot of RNA purification from body fluids, including plasma and serum. Since we are only interested in the cell-free RNA, I have stayed away from PAXgene so I can't say whether or not it interferes with any of the downstream processes. Because I want only extracellular RNA, I do two spins of the plasma: one at 1600× g for 10 min at 4°C to pellet the blood cells, then a second at 16,000× g for 10 min at 4°C to remove cellular debris. Then I use TRIzol LS following the manufacturer's protocol through the chloroform extraction. I purify the RNA from the aqueous phase using either the Ambion mirVana kit or the Qiagen miRNeasy Mini kit following the instructions for total RNA. We don't see a lot of large intact mRNA in plasma, but on a molecular basis (instead of weight), there is a lot of miRNA. I know of others who have found measurable amounts of specific mRNA by qPCR in RNA purified from plasma, but that was shown to reside mostly in the debris, so you might not want to do the second high-speed spin if this is of interest to you.
Looking at your yield from 525 µL of PAXgene plasma, I'm surprised at how much you got, especially since the plasma has already been diluted with PAXgene. This suggests to me that you might be reading absorbance from a contaminant or that you are getting significant cell lysis and the PAXgene is preserving that RNA in the plasma fraction. If the latter is the case then you might actually see some larger mRNA molecules from blood cells.