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Sensitivity of RT-PCR and qPCR


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#1 wdyeo

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Posted 26 May 2013 - 01:50 PM

Dear All,
I inject GFP stem cells into a normal mouse and i harvested the tissues (such as pancreas, liver and spleen) in order to detect if the GFP+ cells are successfully transplanted/home into the mouse.
Is it possible for me to do RT-PCR or qPCR to detect the level of GFP+ cells? Which is more sensitive because maybe only 1 or 2 GFP+cells in whole tissues..Or any other technique besides immunohistochemistry?
I have no idea if the GFP+ cells are transplanted successfully into the mouse by the way :P
Thank you.

#2 bob1

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Posted 27 May 2013 - 01:55 PM

qPCR may work, but the difficulty will be that you will be using so much tissue to get your few cells that the chance of there being transcript off one or two cells in the cDNA prep is relatively low. The only way to know for sure is to get some normal tissue and spike it with a few cells that you have growing, and use that for RNA extraction etc.

#3 wdyeo

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Posted 29 May 2013 - 08:45 AM

Hi,
Thanks alot for the suggestion but I am confuse, how do i take the harvested tissues and spike them on the normal tissues/cells?
Thank you.

#4 bob1

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Posted 29 May 2013 - 03:18 PM

Take a normal liver, homogenize and add a known quantity of cells, then proceed with the RNA extraction.

#5 wdyeo

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Posted 04 June 2013 - 12:37 AM

Oh...
okok..thanks alot ya ! :D




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