real-time pcr optimization problem - (Dec/13/2005 )
Hello!
I have a problem with optimizing this one primer. The gene is supposedly to be abundant since in immunostaining it's protein serve as a marker for the particular cell. However, I didn;t get any response until the 28-30th cycle!The melt curve shows that there is indeed higher primer dimer then products.
Theoretically, since it is suppose to be abundant shouldn;t I get a response in the earlier cycles?
I have also looked through mRNA structure through the MZuker software and the structure was very tightknit and full of crosslinkages.
Can anyone think of a solution to this problem?Is the problem in the RT step or the pcr step?
SOS!HELP NEEDED ASAP! 
OK..two things to consider here
one, if your RNA-of-interest has a lot of secondary structure, make sure you do the 'melt' step when you are setting up your RT reaction, and don't skimp on the time or temperature
two, if you have lots of primer-dimer, the amount of template present really doesn't matter; the reaction will not proceed properly. you need to either change primers or do more optimization steps if you have not been exhaustive
good luck!
one, if your RNA-of-interest has a lot of secondary structure, make sure you do the 'melt' step when you are setting up your RT reaction, and don't skimp on the time or temperature
two, if you have lots of primer-dimer, the amount of template present really doesn't matter; the reaction will not proceed properly. you need to either change primers or do more optimization steps if you have not been exhaustive
good luck!
Thanks!I will try to follow your advice with the "melt"!I am though looking through for polymerase that works at 60 C or above for my RT. Do you think that will be helpful since the ones im using now is Superscript III from invitrogen?
well, do you mean your reverse transcriptase?
You don't do the melt step with the enzyme in the tube...
I do not know if this is applicable to your protocol, I use M-MLV
you add your primer, template, and a little dH2O in the tube, heat up to 75 for 10', then do a very quick spin and put on ice...this melts secondary structure; if you put it on ice IMMEDIATELY, then the secondary structure is not likely to reform that quickly...then you follow with a cocktail of your enzyme, dNTP's, etc and incubate at 42 degrees.
There must be a similar melt step that you can use with a high-temp reverse transcriptase?
Also, you still need to address the primer-dimer issue. do not take that lightly or it will cost you much frustration in the long run!
I hope you can find a good method
yeah i tried what you suggested today..let's hope it works!^_^
*finger's crossed*