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qRT-PCR Product Size - (Jan/21/2010 )

Hi Everyone,

I should be getting a very robust increase in mRNA expression for a certain gene with my treatment, however I'm seeing only a negligible change. I suspect the primers are a problem (rather than my treatment) because I get the expected results when I look at other genes of interest. The set of primers I am using give a product size of about 350bp. I'm hearing that optimal product size for qRT-PCR should be about 150bp.

I doubt this is the case, but do you think the larger product size could be masking my results by reducing efficiency? (I get a C(t) of about 23 with these primers).

Also, a small bump always comes up during the melting curve just before the peak (about 1/4 the size of the main peak) with these primers. When I run my sample on a gel, I don't see any extra bands...just one band at my expected size of 350bp. Could this "bump" also be a problem?

Thanks for any suggestions or ideas!

-rustyshackleford-

Hi rustyshackleford,

In my experience, I don't think that qRT-PCR would contribute much to your current problem, for as long as the primers are well design with great PCR efficiency and there is no primer dimer. Sometimes, I do need to design several sets of primers for the target and then empirically determine which primer set that gave me the best result.

Would you mind uploading the melting curve that shows "the small bump"? I am not very sure if this small bump you are referring to is primer dimer or not?

And is the small bump present only in your NTC, or in all the other sample as well?

-stylothecancer-

Hi stylothecancer,

Thank you for your reply!

I have attached a melting curve showing the small "bump". This bump is never present in my negative control (which shows up as a flat line). It always shows up in my samples which are derived from either animal tissue or cultured cells. When I use other primers on the same set of samples to examine the expression of a different gene, the melting curve is "normal" (i.e. no bump - see 2nd attachment).

Thanks again for your help!

-rusty
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-rustyshackleford-

Hi rustyshackleford,

After looking at your result, some questions come to my mind, and they are:
have you optimised the primer concentration?
have you optimised the magnesium chloride concentration? Or are you using master mix?
what is you PCR efficiency?

-stylothecancer-

Hello again stylothecancer,

I have not optimised the primer concentration. I have always just used 5 pmol per 20 uL reaction (or a final primer concentration of 250 nM, if my calculations are correct). Do you think I may be using too much primer and getting primer-dimers?

I also have not optimised the magnesium chloride concentration as I am using a master mix.

My average PCR efficiency using these primers appears to be around 120%.

Thanks in advance for you time!

--rusty

-rustyshackleford-

Hi rustyshackleford,

The primer concentration that you use should be ok. But is great that if you try to do primer primer concentration optimization to see if the primer efficiency can be enhanced or not as in my opinion PCR efficiency of 120% could be a bit high. =) Normally i will try to optimize the concentration within this final concentration range 50300 nM.

-stylothecancer-