Housekeeping gene: actin

Alex Dobrovic adobrovic at medicine.adelaide.edu.au
Wed Nov 12 16:59:56 EST 1997


Whereas B-actin and GAPDH are useful controls for Northerns, they are close
to useless for RT-PCR because of the presence of numerous pseudogenes in
humans and presumably other species. Run a RT negative control with just
about any primers and you still will get a signal.

Controls that you could consider are housekeeping genes without pseudogenes
such as abl or B2-microglobulin though the presence of pseudogenes will
vary from species to species.

For human work see  Kidd V, 1997   Debate round-table. Appropriate controls
for RT-PCR.  Leukemia 11(6), 871-881 (1997)



>     Z. Huang (huangz at hotmail.com) wrote: : Hi, bionetters,
>
>     : Now I am looking for primers for house keeping genes such as beta
>     actin or : GAPDH in rat and guinea pig. However, in case of guinea pig
>     I can only find : a short fragment of cDNA encoding GAPDH from
>     genebank. And it's even worse
>     : that I can't find published sequence data for rat beta actin. My
>     problem is: : I want to design a set of primer to amplify cDNA of
>     above house-keeping gene : from rat, guinea pig. If it's possible this
>     set of primers can also be used : on human and rabbit. Is there anyone
>     who know such a "universal primers for : house keeping gene"? I would
>     be very appreciate to any suggestions. Thanks.
>
>
>     Huang,
>     I cant find your original posting, so i'm not sure of your original
>     question but hope this helps...
>     I thought actin had been sequenced in all species, but there you go!
>     If you want to use b-actin, any exonic b-actin sequence you can find
>     in any species will work in your system because of the high degree of
>     conservation: human and yeast beta actins are still 98% identical in
>     coding sequences, such is the selective pressure....
>     Generally actin is a good because it can be used as an "all-species"
>     control but be aware that levels of non-muscle actins can vary in some
>     conditions (eg low serum)...
>     Personally i have been using 18S rRNA as a control, see previous
>     recent discussion at this site...
>     Deb

Alexander Dobrovic, Ph.D.
Chief Scientist
Department of Haematology-Oncology
The Queen Elizabeth Hospital
Woodville, SA 5011, Australia

Affiliate Senior Lecturer
Department of Medicine
University of Adelaide

Tel 61-8-8222 6884
Fax 61-8-8222 6046





More information about the Methods mailing list