Internal Standards for RT-pCR - (Jun/25/2008 )
QUOTE (Ned Land @ Jul 1 2008, 10:29 AM)
you can order the primers of about 12 HKG (sequences are published), run them with sybr green and analyze with the free geNorm software to find the two most stable HKG and then normalize against them (or even more).
Thanks for the info, never knew about geNorm. I am working with PIG so I dont have many published choices, however interesting info.
-pcrmossad-
QUOTE (littleaxt @ Jun 26 2008, 12:25 PM)
Hi
If you have the oportunity to control for the amount of cells you extract RNA from you could also spike the reaction with an artifical standard like firefly luciferase or what ever you like. If you can't control for the amount of cells you can normalize against the same amount of total RNA used for the RT, but this is a bit vague and less accurat as the RNA proportions between cells may vary.
If you search for another HKG you can have a look here: http://medgen.ugent.be/rtprimerdb/ or http://web.ncifcrf.gov/rtp/gel/primerdb/
Lots of information you can find also here: http://normalisation.gene-quantification.info/
Cheers and good luck!
If you have the oportunity to control for the amount of cells you extract RNA from you could also spike the reaction with an artifical standard like firefly luciferase or what ever you like. If you can't control for the amount of cells you can normalize against the same amount of total RNA used for the RT, but this is a bit vague and less accurat as the RNA proportions between cells may vary.
If you search for another HKG you can have a look here: http://medgen.ugent.be/rtprimerdb/ or http://web.ncifcrf.gov/rtp/gel/primerdb/
Lots of information you can find also here: http://normalisation.gene-quantification.info/
Cheers and good luck!
Thanks a lot, Actually t seems that there is a paper specifically on Porcine -muscle, reference / internal control genes. Thanks again!
-pcrmossad-