Hello
I am working on Human regulatory T cells. I culture PBMCs and stimulate them. After that, I isolate subpopulation of regulatory T cells using MACS technique and then perform syber green qReal Time PCR assay for my target genes.
The problem is RNA isolation and cDNA synthesis from MACS sorted cells since I have low number of cells (less than 25 000).
Which RNA extraction and cDNA synthesis kit is appropriate for low cell numbers?
I also read about cell to cDNA or Cell to C-T kits. Is anyone familiar with them? I want to know If anyone has worked with them, and do they really work for small number of cells? (since available data are regarding different cell lines, might they be good choices for regulatory T cells?)
And, any other choices?
Thanks to all,
Maryam
I worked on PBMCs culture and used the following kits for RNA extraction and cDNA synthesis and they worked fine..
https://www.thermofisher.com/order/catalog/product/12183018A
https://www.thermofisher.com/order/catalog/product/4387406
We regularely MACS sort. Afterwards Trizol LS with Glycogen in the precipitation step, RT Maxima from Thermo or AffinityScript from Agilent. RT Maxima works still a bit better.
Good luck.
I had the same issue when I started to isolate DCs. I also obtain RNA from 25000 cells and RNeasy Micro kit (Qiagen) worked well for me. It is enough material for few RT-PCR.
Hope it helps.
I would also recommend Rneasy Micro Kit. I used it to extract RNA from polysaccharide scaffold loaded with cells. the cell number was not so high, moreover polysaccharide used to entrap RNA. so to increase the yield, I used to use glycogen or RNA carrier. cDNA making does not require a lot. Also during elution step I used volume smaller than the volume recommended to concentrate my RNA. For cDNA making, I use Transcriptor First Strand cDNA Synthesis Kit (Roche). it works very well so far. For my genes, I have both Randon Hexamer and Anchored Oligo(dT)N primers and both worked very well and show same results on gel electrophoresis.
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