Housekeeping genes for murine lymphocytes

3 posts / 0 new
Last post
piskie
piskie's picture
Housekeeping genes for murine lymphocytes

Hi,
I'm just starting work on Real-time PCR and have to find a housekeeping gene/genes that are suitable for use as a standard in analysis of IGF2 expression in activated lymphocytes. I understand that I need a gene whose expression is not changed during the process of activation. Could anyone give me any suggestions?
Thanks.

Grebo
Grebo's picture
piskie wrote:Hi,

piskie wrote:

Hi,
I'm just starting work on Real-time PCR and have to find a housekeeping gene/genes that are suitable for use as a standard in analysis of IGF2 expression in activated lymphocytes. I understand that I need a gene whose expression is not changed during the process of activation. Could anyone give me any suggestions?
Thanks.

What system are you using?(TaqMan or Sybergreen)

If TaqMan use 18S endogeneous control set,
it has only one exon (cant detect gDNA contamination) but just say to yourself, everyone else uses it and it is good enough for the rest of the community and leave it at that. If not be prepared to eneter a world of unknowns
e.g pseudogenes, DNase I treatment (random cuts do not = gDNA elimination!), and extraction protocol influence on signal
(Do column chemistry Isolation methods fagment the RNA?)
Do everything you can to ensure that you have clean mRNA before RT rxns and you will be fine. What I recommend is to take your Total RNA isolates and do a rxn with reverse transcriptase(RT) and one with out RT, Then do a traditional PCR, run a gel and have a lookie loo. Anything amplifing in the -RT rxn. means you have gDNA contamination and either further purify or throw out. Paste the image of your gel in the lab book and your golden.
If you use sybergreen you can just do a melt analysis of the product but this is where 18S fails, One exon = no distinction of gDNA from cDNA) You could alternatively use GAPDH or Beta Actin. These have exons but they also have pseudogenes that can be( and are ) transcribed.

I attached two excel spread sheets. One is a of a list of House Keeping genes that lists the more popular ones and Assession numbers. And here is a link to the paper from which it is derived;
HKG
And another of known pseudoe genes to various HKGs.
If you need help on the design of your own endogeneous control primer set let me know. Some simple considerations to keep in mind otherwise it is the same as any other PCR reaction.

And here are two sites with good information of the topic of pseudogenes...
mad-cow.org and pseudogenes.org

ToNoFo
ToNoFo's picture
lots og good info in the last

lots og good info in the last post. I personally use beta-2-microglobulin as GAPDH and actin can vary if you use strong inflammatory stimuli (although some may beg to differ). Another possiblity is using Pol II, although I can't comment as I haven't used it. Ironically, the best "control" is really high quality RNA adjusted to equal concentrations. If you have done this, then any variation between samples should be less than 2 fold.