mRNA level in transgene cell line or knocking down cell line - (Sep/08/2009 )
Recently I overexpression a gene and knocking down the same gene in 3T3-L1 cells.
I check the mRNA level of this gene
I found the mRNA level of this gene in overexpression cell line did not go higher, just 1.1 folder higher than control. Is It possible?
I use retrovirus for infecting the 3T3-L1 cells and select the overexpression clones.
Also as for the knock down this gene by siRNA, I check two primer pairs,
one primer pair show the mRNA level in knocking down cells was 70% of control.
however another primer pair shows that mRNA level in knocking down cells was 30% of control.
Why the same cDNA but different primer giving me different knock down efficiency. 70% vs 30%, which one I should believe?
Thanks
ucsd on Sep 8 2009, 02:17 PM said:
I check the mRNA level of this gene
I found the mRNA level of this gene in overexpression cell line did not go higher, just 1.1 folder higher than control. Is It possible?
I use retrovirus for infecting the 3T3-L1 cells and select the overexpression clones.
Also as for the knock down this gene by siRNA, I check two primer pairs,
one primer pair show the mRNA level in knocking down cells was 70% of control.
however another primer pair shows that mRNA level in knocking down cells was 30% of control.
Why the same cDNA but different primer giving me different knock down efficiency. 70% vs 30%, which one I should believe?
Thanks
I would say that your overexpression line did not work.
To troubleshoot:
What kind of retroviral vector are you using?
Does it have an IRES separating your gene and the antibiotic resistance?
Do you have any means of selecting your clones other than mere survival in the antibiotic, like a GFP marker in your plasmid?
Did all of your non-infected cells die in the same antibiotic medium?
Have you successfully used this vector and cell line in the past?
What cell line did you package your virus in?
How did you titer your virus?
Your knockdown line may have worked, but you can't just decide which value to believe regarding the 30% or 70% knockdown
For the knockdown line:
How are these primer pairs different (i.e. is one 5' and one 3')?
Have you checked these primer sets for amplification efficiency?
Are these primer sets validated? Have they been used by others?
Are your primers specific for mRNA (meaning that they won't amplify gDNA)?
Have you checked the protein level? 70% knockdown is pretty good, you should be able to see this easily by Western analysis.
I check the western blot of the overexpression clones, it is fine. Overexpression clones greatly increased the protein level.
I use pbabe-puro vector which did not contain IRES. I use phoenix eco cells for packaging the virus and infecting the cells. I use puromycin for select the clone. For titer, the amount of puromycin I use could kill all the control cells.
For the knock down experiment, I have checked the protein level, It worked. I think maybe primer have problem.
I would say that your overexpression line did not work.
To troubleshoot:
What kind of retroviral vector are you using?
Does it have an IRES separating your gene and the antibiotic resistance?
Do you have any means of selecting your clones other than mere survival in the antibiotic, like a GFP marker in your plasmid?
Did all of your non-infected cells die in the same antibiotic medium?
Have you successfully used this vector and cell line in the past?
What cell line did you package your virus in?
How did you titer your virus?
Your knockdown line may have worked, but you can't just decide which value to believe regarding the 30% or 70% knockdown
For the knockdown line:
How are these primer pairs different (i.e. is one 5' and one 3')?
Have you checked these primer sets for amplification efficiency?
Are these primer sets validated? Have they been used by others?
Are your primers specific for mRNA (meaning that they won't amplify gDNA)?
Have you checked the protein level? 70% knockdown is pretty good, you should be able to see this easily by Western analysis.