Beta-Actin good RT-PCR Control?
Richard R. Hardy
hardy at mighty.fccc.edu
Fri Dec 30 15:31:15 EST 1994
In article <3e1ioo$1nn at netnews.upenn.edu>, smoore at mail1.sas.upenn.edu
(Sean David Moore) wrote:
> Dear BioNetters....
> Here's a situation for you. I have been isolating human alveolar
> macrophages from sick patients. I treat them with different experimental
> proteins and later look for message of cytokines. I have lately had
> trouble finding beta-Actin as a positive RT-PCR control..I think there
> may be two problems:
> 1) We are using total RNA for the RT, not mRNA ( not enough sample )..
> this may drown out the Actin message during Random primed RT...so I
> switched to Oligo-dT...I still worry about using too little actual mRNA
> in the RT.
> 2) Also, these macrophages have benn settled for at least 24 hours
> before treatment. Their beta-Actin production may be at low levels to
> start with and treating them with something like LPS could severely
> reduce Actin message...I may be looking for the wrong positive control...
We routinely use b-actin with cDNA made from total RNA isolated from
10^4-10^5 cells and it usually gives a nice EtBr band after 18-20 cycles
(using 1/5 of the cDNA). This includes resting B cells. We get good RNA
using GITC/CsCl method (the "everybody does it" technique). Recently
we've found that the "TRI-Reagent" (quick prep, no CsCl spin) also gives
good EtBr b-actin signal (at 35 cycles) from as few as 10 cells (!). So I
think it's a good positive control and total RNA should be OK; we do,
however, use random hexamers, not oligo-T. Perhaps your RNA is being
degraded by the macs?
Member, Institute for Cancer Research,
Fox Chase Cancer Center, Philadelphia, PA
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