Hi,
in principle you could use the protocol for single cell as it is, provided that the input RNA is not too high. I don´t know exactly, maybe up to 1 ng. If you start form several ng you should increase the amount of some reagents, to avoid their depletion during the RT or PCR. I would (as an example) double the amount of TSO, ISPCR and oligodT primers and increase also the conc of dNTPs. Again, you should run some kind of titration to see what reagent becomes the limiting factor in your reaction. If planning to sort hundreds of cells in each well, you might also consider using a higher conc of Triton to ensure proper lysis (or a stronger buffer like guadine thiocyanate/hydrochloride, RLT, etc). Concentration of Triton of 1-2% do NOT have a negative effect on the RT reaction. If you need extra info just give me more details about your experiments
in principle you could use the protocol for single cell as it is, provided that the input RNA is not too high. I don´t know exactly, maybe up to 1 ng. If you start form several ng you should increase the amount of some reagents, to avoid their depletion during the RT or PCR. I would (as an example) double the amount of TSO, ISPCR and oligodT primers and increase also the conc of dNTPs. Again, you should run some kind of titration to see what reagent becomes the limiting factor in your reaction. If planning to sort hundreds of cells in each well, you might also consider using a higher conc of Triton to ensure proper lysis (or a stronger buffer like guadine thiocyanate/hydrochloride, RLT, etc). Concentration of Triton of 1-2% do NOT have a negative effect on the RT reaction. If you need extra info just give me more details about your experiments
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