When you working with serum it is unusual to see white pellet considered as RNA sediment at the end of procedure. It is mostly due to very low concentration of cell-free RNA in bio-fluid such as serum. You can testify your extraction quality using Nanodrop. If you have high salts concentration it will appear in absorption curve. If the salts contamination confirmed, then you must make sure that all of your materials are highly-purified, I mean molecular biology grade. You've mentioned that aqueous phase is a bit cloudy but it has to be clear and transparent so it might be a sign of malfunction in previous step. So check your chloroform supply first.

@Darrell Green , EDTA or heparin are used to extract plasma from blood samples not serum! serum separation would take place in sterile test tube or clot activator tubes without any additives . by the way do you have any good reference about using carrier RNA? I've never used that but if it rise RNA yield I really want to give it a try.

I am also using miRNeasy isolation Kit for serum. Are you using the ideal amount of Qiazol for the extraction of 100 microLiter? Because when aqueous layer is cloudy maybe you are not degrading all proteins that are together with miRNAs. There is a QIAGEN Supplementary Protocol for serum that might help you. To known if your sample degradation is sufficient, your sample with Qiazol must be very liquid and not viscous. If after pipeting Qiazol several times you still have a viscous, I suggest to add more Qiazol to the sample. But remember to keep the proportion with cloroform. Best regards!

Try another kit. it's strange.

I have seen this ppt occassionally but my RNA quality was always fine. Ambion claims that some glass particulates can sometimes escape the filter. I'm not confident in their answer but my RIN values were fine and samples have been good for RT-qPCR and NGS so it is possible. It appears to be variable by kit lot which could also support their suggestion.

Thank you all for your comments. Turns out it was a problem with denaturation. I increased the time with vortexing and it worked. I have a clear aqueous layer without white ppts in the end. Now the other problem is yield of miRNAs.

I have 2 questions for now ~

1. Exogenous control concentration ~ I am using cel mir 39 as exogenous control at a concentration of 0.91 fmol/3.5microliters (Dilutions suggested by Qiagen kit). I see people using around 25-30 fmols. What is your inputs on that. Does this also have a role in yield?

2. Carrier for isolation~ Which one is better Glycogen (what conc) or MS2 RNA (Roche)? Any other carrier? We have Yeast tRNA, is it alright to use as it says its for co-precipitation of RNAs.

Would really appreciate your inputs. As the thread is not very old, so posting in here only.

best regards,

Minakshi

Dear Minakshi,

I did a dosis curve for three different cel-miRs for nomalization (starting with 25fM until 250pM) and I used real time PCR to choose. The best Ct in PCR was at 25pM and now I put 5 microliters of each cel-miR in 25pMol in my sample during Qiazol extraction. I sugest you to do a dosis curve in your experimental condition.

Best regards,

Leandra

Dear Leandra,

Thank you very much for the reply. Sounds like a ideal thing to do. If its not too much, could you please share your protocol here or email me on [email protected]