Beta-Actin good RT-PCR Control?

Sean David Moore smoore at mail1.sas.upenn.edu
Fri Dec 30 13:15:52 EST 1994


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...


Any Help would be appreciated.

Thanks in advance.



--
Sean Moore

VAMC Philadelphia,
Medical College of Pennsylvania,
and the University of Pennsylvania...

.

smoore at sas.upenn.edu





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