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Real time house keeping genes change


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#1 ozposdoc

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Posted 01 July 2009 - 10:58 AM

Can someone please help me. I have done real-time validations for my GOI and 3 housekeeping genes. Validation Efficiencies were great. So I ran the experiment - but my housekeeping genes are different between my two groups. However, I tested all 3 housekeeping genes and they change the same way - eg sample 1 for actin and 18s gives me a ct of 20 and 18, respectively, and sample 2 gives me 22 and 20 - so the degree of change is the same.

So my question is - can I normalise somehow since 3 housekeeping genes change in the same degree. The change most probably reflects to template amount (although I did check before RT PCR) - however, instead of repeating the experiment - (don't want to waste more SYBR GREEN) - can I do something with the results I have?

PLEASE HELP ;) and thanks in advance ;)

sad ozpostdoc

#2 cellcounter

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Posted 01 July 2009 - 05:06 PM

Can someone please help me. I have done real-time validations for my GOI and 3 housekeeping genes. Validation Efficiencies were great. So I ran the experiment - but my housekeeping genes are different between my two groups. However, I tested all 3 housekeeping genes and they change the same way - eg sample 1 for actin and 18s gives me a ct of 20 and 18, respectively, and sample 2 gives me 22 and 20 - so the degree of change is the same.

So my question is - can I normalise somehow since 3 housekeeping genes change in the same degree. The change most probably reflects to template amount (although I did check before RT PCR) - however, instead of repeating the experiment - (don't want to waste more SYBR GREEN) - can I do something with the results I have?

PLEASE HELP :( and thanks in advance :)

sad ozpostdoc

Lately, ribosomal protein family has got bigger traction in real-time normalization. Pick up anything starting with RPL (i.e. (RPL19), and it will work better than most other traditional housekeeping genes (b-actin, lamin-b, Gapdh etc). I haven't checked these but look up some protocols on Vadlo, to see if you find some more names.




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