Hi Farah Daou

The isopropanol will also cause polysaccarides and some other components to precipitate, so sometimes when dealing with samples that contain abundant extracellular matrix you will obtain a pellet but have very little RNA. In these cases it is worthwhile to purchase an RNA extraction kit that uses a silica-membrane based spin column. These are much better at removing these impurities. That said, depending on your downstream application 0.1 ug/uL RNA is probably more than enough and there is a good chance that any contaminants won't interfere with subsequent reverse transcription so it never hurts to go ahead and give your downstream application a try. You can ensure you have high quality RNA by running 0.5 - 1.0 ug on an agarose gel with ethidium bromide, this will help you determine how much RNA is actually there and whether it makes sense to proceed.

Hello Zachery R Belak ,

Thank you for taking the time to explain this to me. I have one more question. Do you recommend using the silica-membrane based spin column when dealing with cells that were seeded on bioactive glass or other polymers.

Regards,

Farah

Farah Daou the silica membrane based kits will work fine for that application. The only extra steps would be to ensure that the reagents have adequately penetrated the growth matrix so the cells are well lysed and then to centrifuge the sample before applying the soluble part to the column so no bits of glass etc from the matrix are carried over. I know also that the manufacturers of these kits will provide pretty good technical service and will be able to help you determine if they will work for your application. Also keep in mind that if you are working with very small amounts of cells you may need to reduce the volumes of reagents and the volume of water used to resuspend the RNA so you get a more concentrated sample.

Dear Zachery R Belak,

Thank you for your assistance. I will use TRIzol again this time, but I will centrifuge the mixture before adding chloroform. If this does not work well, I will contact the manufacturer of the kit we have in the lab to know whether columns will work.

Best,

Farah

Dear Farah,

I have the same problem. I have the pellet but nearly no RNA. I am transfecting fibroblast cells with chitosan nanoparticles and then isolate RNA. From the control samples, I can isolate RNA, but from chitosan transfected cells I can not (the interesting thing is that, I have the pellet at the end of trizol protocol but neither QUBIT nor spectrum scan reveals any RNA in the sample). Please someone help, I really appreciate.

Dear Deniz Sezlev ,

How much RNA are you getting when you use the Nanodrop?

I also had a very small amount of RNA (0.1 micrograms in a total volume of 20 microliters), but I was able to run the pcr and get good results. I think that the pellet we are seeing has other particles, as suggested by Zackery (in my case particles of bioactive glass). I also think that we are unfortunately loosing cells in a way or another (in my case, I saw some apoptotic bodies).

I hope someone can help you.

Regards,

Farah

Dear Farah,

Thank you for your response. Here I don't have Nanodrop, I do Qubit reading (with fluorophore binding to RNA) but it gives me "low" value (it doesn't give me any result but I am guessing it is lower than 2 ng/uL; because the sampe concentration is 2 ng/ul it can give me that value). The thing is, this value is even before DNase treatment, and since I don't know the concentration I can not move on to cDNA and PCR. So, from your answer I guess you didn't try anything, right?

Dear Deniz Sezlev

Yes, you need to know the quantity of RNA before moving to cDNA synthesis and PCR. In my case, I used 200 ng of RNA (except for the sample with 100 ng of RNA) to perform cDNA synthesis, and then I used the samples directly for qPCR without prior dilution. However, for the sample with 100 ng of RNA, I added double the amount of cDNA (and decreased the volume of nuclease-free water in the mastermix).

If you have access to a Nanodrop, it may be helpful to check your samples usgin it

I hope this helps.

Regards,

Farah