I need to isolate the RNA from different sub-populations of human PBMCs. From the smallest population, I have cell number varying between 5000-12000 cells. The final end point of the study is to perform miRNA expression array from these sub-populations. Can you please recommend me some protocol or kit which works best with this low number? I tried sorting the cells in Trizol/Trifast but since there is also some amount of sorting buffer (PBS) which is added along with the cells, the original Trizol/trifast is diluted and I get poor RNA in end. I tried to sort cells in PBS/FCS and then spin down the cells and then used Trizol/Trifast or RLT lysis buffer (RNeasy mini kit) but still no success. I know I will loose my miRNA with RNeasy mini kit, but my first part/aim is to get good quality total RNA from this low cell number. Does adding Glycoblue (co-precipitant) will help in Trizol/trifast protocol ? Please help.
All your knowledge and recommendations are highly appreciated.
Regards,
Raghav
I would suggest the spin down of your cells and then follow the RNAeasy protocol. Yes, you won't be able to get miRNA this way, but its a start. Instead of using the RNAeasy column that is normally in the kit, I suggest you use one of the smaller columns that are part of a clean up kit (the ones that allow low elution...5-10uL) so that you can elute your whole sample in a small volume and follow the protocol for the RNAeasy kit....the methodology of binding is the same. Don't worry about "RNAses" in your column as they will be gone during the purification steps from the RLT and RW1 buffers.
Alternatively, the use of glycoBlue will definitely help with the Trizol procedure, and ultimately, should give you small message as well for miRNA
Good luck
Hi Raghav, You might consider a selective precipitation method as an alternative to the spin-column-based adsorption methods. The yields can be much better and the volumes can be scaled as needed to match your sample volume. Keeping the volume small will be helpful for small amounts of material. In my lab we have had very good luck with the MasterPure kit (Epicentre), a protocol modified from Miller et al. 1988 NAR 16(3): 1215. A co-precipitant is already included in the buffer for that kit. We have used it to purify RNA from viruses and DNA from bacteria with good yields and no interference in subsequent enzyme reactions. This includes successful PCR amplifications using template DNA extracted from only a few tens of thousands of flow-sorted bacteria. You can either pellet the cells and extract, or, if the volume is small enough to allow it, add one volume of 2X extraction buffer to the cell suspension and proceed. For this to work for your goal assumes that miRNA is efficiently pelleted by isopropanol. I don't work with miRNA specifically so not sure about that. However, it sounds like isopropanol is part of the Trizol protocol as well, so I assume that part should be fine. However, I also just read about biases in microRNA recovery by the Trizol protocol so be wary of biases in whatever protocol you choose.
You can try with this kit:
https://norgenbiotek.com/product/single-cell-rna-purification-kit , is designed for a range from single Cell to 2 x 10^5 cells.
I used various kits from Norgen and they always worked very well.
I would try the Qiagen miRNeasy Micro kit. Retains miRNAs and you can elute in small volumes (~12ul). You can also use glycogen as a carrier and there is a slightly modified protocol for low amounts.
https://www.qiagen.com/gb/shop/sample-technologies/rna-sample-technologies/mirna/mirneasy-micro-kit/
Another alternative is the Qiagen Micro kit:
https://www.qiagen.com/sg/shop/sample-technologies/rna-sample-technologies/total-rna/rneasy-micro-kit/#resources
I have been using this for small population of micro-dissected frozen specimens (~1000-5000 cells) and was able to obtain good RNA yield with decent quality.
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