How many RNA do you extract and how does this look on a gel?

Dörthe Andrea Kesper Hi, from a culture of 5 million cells I only extract about 0.051 ug/ul. I haven't applied this to a gel. I have just extracted it with a silica column kit and quantified the RNA extracted with a nanodrop. For more context, I tried extracting RNA from a pellet immediately extracted from blood and got an RNA concentration of 0.23 ug/ul, but when I cultured them for 24 h, I only got 0.051 ug/ul. I didn't stimulate the PBMCs with LPS or PHA.

Hi Sukrit,

It is hard to say how many RNA you have id you only give the concentration of the RNA and not the amount of elution buffer you have used.

I would alway analyse the RNA a gel because you can good readings from the nanodrop bur the RNA can be degraded severly impairing your downstream application - if you have good qulity RNA the amount might be enough for downstream applications

It would be really good to know the kit you have used to isolate the RNA - without this information trouble shooting is more diifficult.

Howeve,r regarding you differences you got from your two differenet Experiments. I assume you have used the same amount of elution buffer.

As I do not now the kit and the protocol you used I can give you only limited amount of advise

1. have you check if your cell are still alive after culture for 24h?

2. You might consider stimulate the cells with PHA

3. Don't overload your column

4. Elute twice with the same elution buffer (but it twice on the gell)

5. Heat the elution buffer (up to 60*C to 70°C

Hope this helps

KR

Hi Dörthe Andrea Kesper Apologies for give the answers in chunks. I am new to this so I may be a bit inefficient at first. So I used a total of 30ul of elution buffer (RNAase free water) to extract my RNA and I am using the Monarch Total RNA Miniprep Kit.

Furthermore, after 24 h cells are still alive but there is significant death (15-20%). Yes, I am considering stimulating the cells with PHA or LPS. Which one would you suggest if I want broad activation of the cells?

Thanks for the help, I really appreciate it!

Hi Sukrit,

when I see it correctly you got 1,5 µg of RNA from your cultivated PBMC which is not too bad. The amount you got from you freshly isolated sound rather high.

I have worked with the Monarch Miniprep-Kit before and I rechecked the manual. According to the manual the maximum input amount for PBMC is 1x10^6 cells.

This means you have 5 times more cells as the recommended maximum amount. If you exceed the maximum amount of cells this can

1. lead to an overload of the column and this will severly decrease the RNA yield

2. can lead to contamination with genomic DNA (and I must admit I think this can be the case for you freshly isolated PBMC as the amount of isolated nucleic acid is high)

Overloading the column is a common mistake but this should be avoided (see above).

So I strongly recommend to reduce your cell number - you might get a higher yield of total RNA of better quality

Check your RNA on a gel (100 ng of RNA on a 1% agarose gel should be enough) - here you can see if your RNA is intact and if you have genomic DNA contamination (at least if the genomic DNA is intact).

Check the manual how to get rid of DNA contamination (except from reducing the cell number) if you suspect or see that you have contamination.

Regarding the stimulation PHA might be better for broad activation - however PHA as well as LPS will change the gene expression profile. You might check out this publication (deals with human and monkey PBMCs and compares gene expression between PHA and LPS stimulation):

https://atm.amegroups.org/article/view/60245/pdf

I hope this information will help you with your experiments.

KR Dörthe

Hi Dörthe Andrea Kesper Thank you so much for your input. It was truly helpful and insightful. I conducted a qPCR with the extracted RNA and the results were satisfactory!

Hi Sukrit,

happy to hear that it worked

KR Dörthe