Whole genome amplification from cDNA? - (Jul/04/2013 )
My lab has been trying to amplify cDNA from single cells, through PCR. We've been having assorted ongoing problems, and now they are coming to me with a new protocol, but I'm not sure if this makes any sense. I am a new tech, and no one in my lab has any background in molecular biology, so any help is appreciated.
They want to take the RNA from the single cell, reverse transcribe it into cDNA, then use Qiagen kit REPLI-g, for whole genome amplification of single cells. Does this make sense to anyone? Are there any pitfalls that I should be aware of?
sweetstef on Thu Jul 4 12:03:58 2013 said:
My lab has been trying to amplify cDNA from single cells, through PCR. We've been having assorted ongoing problems, and now they are coming to me with a new protocol, but I'm not sure if this makes any sense. I am a new tech, and no one in my lab has any background in molecular biology, so any help is appreciated.
They want to take the RNA from the single cell, reverse transcribe it into cDNA, then use Qiagen kit REPLI-g, for whole genome amplification of single cells. Does this make sense to anyone? Are there any pitfalls that I should be aware of?
If I understand you correct you want to sequence the genome? Or ?
Sorry if I wasn't specific. Our end result is not sequencing, we are really just looking to see if certain genes are expressed. We aren't doing qPCR because we don't care about expression levels, just whether or not certain genes are there.
sweetstef on Thu Jul 4 12:43:09 2013 said:
Sorry if I wasn't specific. Our end result is not sequencing, we are really just looking to see if certain genes are expressed. We aren't doing qPCR because we don't care about expression levels, just whether or not certain genes are there.
Ah ok, I understand it why they do it. By working with mRNA they know its expressed (at least at rna level). So yes its a common technique to use.
Great, thank you very much.