Hi all,
I am currently conducting an experiment which involves FACS-sorting a specific population of cells using an antibody of interest.
In order to validate the type of cells I have collected using this marker, I want to perform qPCR on their extracted RNA, using certain known cell-type marker primers.
My problem is that in each sorting session, I am only able to collect anywhere from 5,000 - 10,000 cells. Do you know if it is possible to perform RT-qPCR on such a small sample size?
I have collected the cells in 1mL of Trizol.
Thanks for your help.
You will absolutely have success with your qRT-PCR. If these are human cells, 5,000 to 10,000 cells (at 20 pg of total RNA per cell; 15 pg/cell figuring in losses) would give you (after figuring in 17% loss during Trizol extraction) 63-125 ng of total RNA. If the final pellet you obtain after Trizol RNA extraction is solubilized in 20 uL of nuclease-free water, then diluted 1:10 with 1 uL RNAseOUT and 179 uL of nuclease-free water, followed by TURBO DNAse treatment of 80 uL of each of these samples (80 uL sample, 10 uL 10X TURBO DNAse buffer, 10 uL TURBO DNase enzyme for 30 min 37C, followed by addition of 10 uL inactivation reagent and subsequent perturbation for 2 min followed by centrifuging for 3 min at 10,000 g, followed by removal of the top 80 uL of each DNAse rxn), you will then have 80 uL DNased RNA samples that contain ~0.23 to 0.45 ng total RNA/uL. If you then use 4.8 uL of sample per each 20 uL qRT-PCReaction, you would then have reactions that contain ~1.08 to 2.17 ng total RNA per qRT-PCReaction. This amounts to ~54 to 108 cells' worth of total RNA per qRT-PCR - which is more than enough to give you good signal by qRT-PCR. I have loaded as little as 1 cell's worth of RNA in qRT-PCReactions and have gotten great signal this way. One only needs more than 5 transcripts to get a good qRT-PCR signal with high-quality, non-inhibitory, DNAse-treated RNA isolates. This is a "one-step" qRT-PCR I'm referring to: quantitative Reverse Transcription Polymerase Chain Reaction; wherein you load the above prepared RNA sample into a two-enzyme (both Reverse Transcriptase and 'Taq' DNA-dependent DNA polymerase enzymes present) master mix + primers or primers and probe. It always helps to calculate how many cells' worth of material you are loading per each final qRT-PCR... And, Trizol is a great way to go for this: you won't ever see a pellet - but it is there~! So just keep on working forward all the way through the qRT-PCR and you will be fine. You won't be able to get Spec or NanoDrop readings on the samples either - but, do not let that stop you from moving forward - the qRT-PCR result will be just fine. Also: do not sneeze!
In addition, I'd like to suggest you to add 3-5 ul of molecular biology grade Ultrapure glycogen (I use a glycogen supplied at 20 ug/ul) for those low-quantity samples at the isopropanol precipitation step.
This will increase the yield of RNA during precipitation and eventually you'll be able to see the position of glycogen pellet and obviously RNA pellet will be inside of it. This will help you when you'll resuspend the pellet in DEPC water.
If you are worried about Trizol extraction (or have problems extracting RNA from your cells with it) - I have read about a kit that can extract RNA from small amounts (up to 5mg of tissue or small number of cells [up to 5 x 10^5) such as laser-captured cells, microdissected cryosections, fine needle aspirates, biopsy samples, cultured cell pellets and flow cytometer sorted cells). I know kits can be different from country to country, the one I was thinking about is called ISOLATE II RNA Micro Kit (http://www.bioline.com/de/isolate-ii-rna-micro-kit.html). I haven't tried the Micro Kit myself yet, but the Mini kit from the same range worked very well with certain samples. Good luck for your work.
Its good to collect the cells into Facs Buffer. After collecting the cells, spin it for 300g for 10 minutes. , Remove the sup by tipping it off then add 500uL of trizole. Do not add such a high volume of 1ml Trizole....
Paolo is right..... Add Glycogen along with isopropanol precipitation.... You will get RNA for cDNA preparation....
If this technique don not work well then you may try cell to ct Kit
Good Luck
In laboratory I previously worked we had good experiences after amplification and conversion of RNA to cDNA using Nugen Pico kit.
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