A student in my department has recently submitted some RNA samples to a sequencing centre for NGS. He was advised that there were problems with the library prep due to vicosity of the RNA samples. All the usual QC was run prior to submitting the samples (260/280, 260/230, gDNA contamination, Qubit conc. and Bionalayzer to check RNA integrity) and samples all checked out within the usual acceptable standards. The seq centre has suggested that the viscosity is due to high gDNA contamination but we found this to be less than 10% across all the samples.
Has anyone ever encountered this problem before? If so do you have any suggestions as to how to overcome it? Any and all suggestions welcome!
Hi Nicola...
This issue may be related to sample type rather than the extraction protocol!!
Try this link...hopefully it will be helpful.
Best Wishes.
Article RNA Isolation from highly viscous samples Rich in Polyphenol...
Thanks for your reply Ayoob :) This was my first thought also - however the student has submitted RNA extractions from this tissue type before and library prep went ahead and good read data obtained. It seems that the samples were "viscous" on that occasion too but this time it seems to have caused a greater problem. I had a look at the link you sent and it seems to be dealing with a problem where the tissue itself is viscous. We find that the tissue being used here is not viscous or difficult to extract from and there are no obvious problems during the extraction process. We really are a bit stumped on this one!
It could be because of carbohydrates. Especially charged ones, like oligosaccharide phosphates. They do not absorb at 260/280 but contribute to sample viscosity and may affect RNA solubility when present at high concentration. It's hard to get rid of them by precipitations or extractions or using spin-columns. The best way to remove them is to run sample on a hydrophobic-interactions column (like Phenyl Sepharose or so). Gel-filtration can be useful too.
10% contamination with gDNA will easily give you viscous samples. You can enzymatically eliminate the gDNA with any number of DNase/column purification kits. You could also just shear the samples with narrow gauge syringes, but it's probably better to get rid of it.
I used to do quite a lot of RNAi in Drosophila embryos. When knocking out more than one gene at a time, I frequently doubled and quadrupled the recommended dsRNA concentrations for injections. The 4 genes at a time was the most we could do, because the viscosity got to be limiting---- we couldn't ejects through a pulled pipette at concentrations higher than this. So i think RNA (admittedly in this case it was double stranded) can be quite viscous and you jsut need to measure your concentrations carefully. I suspect your student just did a final resuspension in too small a volume. I looked back at these posts in this thread and can't find what the UV spec readings were, so I don't really know what they measured. But I would just go back and check concentration.
Ideally the final RNA pellet after dissolving in TE/DEPC water will be very clear. I would face this problem when extracting RNA using Trizol reagent, I would dry the pellet after the ethanol precipitation step. If the pellet was over dried then the pellet would not dissolve and would result in the viscous solution. So I reduce the drying time of the pellet and also ensured that the ethanol was evaporated and then dissolved the pellet.
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