Hi, can I ask what type of non coding RNAs will you study?

I don't understand very well the part about "plasma ....diluted with a physiological serum". Do you mean that the plasma was obtained after the PBMC extraction so the blood was, initially, diluted with PBS?

Valeria De Arcangelis

Dear scientist

Thank you so much for your reply.

I'm studing on circulating Long non coding RNAs (LNCs).

Yes, the plasma was obtained after the PBMC extraction so the whole blood was, initially, diluted with sodium chloride 0.9%.

Hi, I have experience with microRNAs not with long non coding but there are some similarities. I personally use kit for microrNAs extraction because I decide to use Qiagen microarrays so I followed the suggested procedure.

I found this article

https://pdfs.semanticscholar.org/9317/4d158f37debe5be7e21b78bc61f5b7cbdd3c.pdf

They optimized some step during extraction for example using glicogen and 3 mM Na-acetate for better precipitation.

With microRNA, but also with other non coding RNAs, can happen to have low A260/280 ratio for total RNA because it is difficult to read with nanodrp from this type of samples. In most case some kit, used to enriched small non coding extration, suggest to not read with nanodrop. You use trizol so you want to read total RNA extraction and I understand that you want to be sue tha tyou have good RNAs, but with plasma samples can be a challenge.

In your case I thought about a possible contamination not only for the 1,6 ratio but, in particular, for the source of plasma. You have dilution and also Ficol it is not an ideal situation. All the pplasma /serum extraction were peformed from centrifugation from fresh blood directly, or from PBMC without the blood dilution (it is possible, in your lab we try it). Another thing it is that your collected the blood in heparin tubes and it is not the best thing for further PCR analysis, because heparin can inhibit Taq pol.

I don't want discourage you but only said thet can be present other aspects that make worse your RNA extraction.

From my point of view first you have to ptimezed the protocol without wasting your precious samples so use fresh blood from a volunteer (you, your co-workers), procede with the protocol, use the articles tips, read you A260/280 ratio, make a gel. You have to be sure that you can make a good RNA extraction from plasma!

Then try again with you stored samples bearing in mind that you have samples stored to -20°C for so long (it would be better -80°C). You don't have "pure" plasma, but plasma mixed with Ficol, platelets, debris 8remember to centrifuge before putting trizol). How much volume of diluted plasma you have at the end of Ficol procedure? Depending how much blood you have initially (at least 6-7 ml) and what type of dlution 1:1-1:2, after Ficol centrifugation, you can have a lot of volume above the PBMC ring and this is all the plasma diluted. How much trizol do you use?

If you procede to extract from "not diluted" plasma and obtain a good results I am sure you will understand the problems with the stored "diluted" plasma that you have to analyse.

Valeria De Arcangelis

Hi Dear Dr. Valeria De Arcangelis

I should like to express my sincere thanks for your valuable advice and all the help you have given me. please kindly accept my apologies for the delayed response.

Would you please mention other ways for evaluating quantity and quality of total RNA extracted from plasma rather than using Nonodrp and gel?

That's a great idea to use fresh blood from a volunteer and I 'll do it; mean while I have done this procedure with pure plasma but the yield was 123 ng/ul and A260/280 was 1.47.

In this protocol,first whole blood was diluted with sodium choloride 0.9% and the volume of blood, sodium choloride and ficoll was equal, it means the plasma is diluted 1:2.

In trizol protocol, generally 250µl plasma/750µl trizol should be used, but as I mentioned the plasma has been diluted therefore I used 500 µl plasma/750 µl trizol.

Sincerely

Hi, first of all to answer you question, in the article sometimes use more sophisticated instrument as bioanalyzer, I don't have experience with this.

In general now is more simple to use extraction kit optimezed on plasma/serum so you have an enrichement in small RNAs, there are kit for microRNAs or long non coding and generally you don't read the RNA but follow the protocol for the further step (RT, preamplification and microarrays).

With RNA in body fluid it is really difficult the quality/quantity check with the normal instrument in lab. A lot of people on researhgate point out that have low A260/280 and A260/230 ratio. You have to work with difficult samples I am sorry!

For the part about your RNA extraction with non diluted plasma, did you analysed also the A260/230 ratio? If you have problem with extraction in non diluted plasma you have to understand if you have contamination (A260/230 ratio ratio can help) or degradation of RNA. You have to solve this part to be sure that in your hand all work fine.

In the last part you said that you use 500 µl plasma/750 µl trizol, but I would the protocol 1:3 (sample:trizol). I known that you have diluted samples but you have PBS, Ficoll, salt maybe other contamination from Ficoll so I would try more Trizol. if is a tube problem try to split in two tubes (respect 250/750), and mix again as soon as possible. I recommend sterile glicogen.

NB: Important reminder for further step

1The microRNAs are different from long noncoding but remember that in body fluid the normalization is a big issues respect the cells or tissue samples. I don't known if you need spike in the long non coding analysis but, check information about long non coding normalization in plasma or you can have problem later

2 Even if you will use simple real time reaction or a custom microarrays perform always the preamplification step, the small RNAs are low in the plasma /serum (in your case less in healthy than in cancer patients) and you risk that you don't see any amplification. I made this mistake once.