How much RNA typically we can get RNA from any single cell? Has anyone isolated RNA from single cells? Using which method or protocol? Based on your experience, what possible downstream assays can be done after single cells RNA isolation?
Thank you.
Hi Kamlesh
I usually get 3750-4000ng of total RNA from 150 000 cells (HK2 cells). So, per cell it’s around 0.025ng (25pg). This number can be slightly higher or lower based on the cell type.
Use PicoPure™ RNA Isolation Kit (Thermo Fisher Scientific). It can be used to isolate RNA from 1-10 cells.
Good Luck..!
Susara
Hi Kamlesh
I usually get 3750-4000ng of total RNA from 150 000 cells (HK2 cells). So, per cell it’s around 0.025ng (25pg). This number can be slightly higher or lower based on the cell type.
Use PicoPure™ RNA Isolation Kit (Thermo Fisher Scientific). It can be used to isolate RNA from 1-10 cells.
Good Luck..!
Susara
Hello Susara,
Thank you for adding answer. Have you used PicoPure Kit? Does it produce reliable results?
Thank you.
Hi,
Depends on the cell type, you get dozens of picograms of total RNA.
The downstream could be various - RT-PCR (Takara ICELL8, etc), scRNA-seq (Takara ICELL8, Fluidigm C1, Dolomite Nadia, 10x Genomics, etc), scATAC-seq (Takara ICELL8, Fluidigm C1).
Anyway, due to the low RNA concentrations, most of the procedures are made in one go, without any extractions or cleanups. For example, for scRNA-seq usually you do lysis, RT, unique barcoding and cDNA amplification in one step. You only do a cleanup of pooled cDNAs from many cells before the final library preparation.
Best,
Michail
Thank you Michail, Thats helpful information. I will look those keywords you mentioned regarding downstream applications.
Hi Kamlesh,
I'm so sorry that I haven't used this kit for my own experiments. But our previous lab used it and had no issues.
Good Luck..!
Susara
hi
we usually calculate that a typical mammalian cell contains 10–30 pg total RNA.
The majority of RNA molecules are tRNAs and rRNAs. mRNA accounts for only 1–5% of the total cellular RNA although the actual amount depends on the cell type and physiological state. Approximately 360,000 mRNA molecules are present in a single mammalian cell.
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