Starting real time rt-pcr - working with rna virus (Feb/03/2006 )
Hi! I have really big problems with my real time rt-pcr. I´m working with an RNA virus so, my real time has to be rt-pcr. I use SYBERGREEN but have no results. My protocol is :
12,5 master mix ( stratagene)
0,25 each primer at 20mM conc.
5 microlitres of RNA extracte with a silica filter method.
0.0625 Rnase Inhibitor
water until 25microlitres
I have no amplification at all, only primer dimer but in conventional RT-PCR it works really well. My template is 300bp, I think it´s a good lenght.
Please, need some help!
I do the RT in one step but RT enzime is included in the master mix.
My program is:
30 min 48ºC (RT)
10 min 95ºC (RT inactivation)
40 cycles of 45sec 95ºC-2min 60ºC
7min 72ºC
At the end, I read the fluorescence to make the dissocation curve: 95ºC for 1min and an increase of 1ºC each 30sec (reading fluorescence)
I'm confused, are you doing one-step? where is your reverse transcription step? is it included?
First step is to transcride your RNA to cDNA.....I cannt see it in your protocol. Elaborate on this please
He is doing one step. That is what the 30 minute step at 48 degrees celsius is for.
I would never do it that way, however.
Two-step is preferable.
-Matt
hey, Matt, he added his protocol in an edit after I responded...I know you don't do PCR at 48C 
So, I would like to ask if you have done anything to optimize? such as, adjusting primer annealing temp and so forth?
And, except for the master mix, how does this protocol differ from what you do when you set up the conventional method?
So your PCR is two-step during the 40 cycles, right? your 7 min at 72 is your final extension step?
and I am guessing you know it's primer-dimer based on the dissociation, or do you run a gel?
So, I would like to ask if you have done anything to optimize? such as, adjusting primer annealing temp and so forth?
And, except for the master mix, how does this protocol differ from what you do when you set up the conventional method?
So your PCR is two-step during the 40 cycles, right? your 7 min at 72 is your final extension step?
and I am guessing you know it's primer-dimer based on the dissociation, or do you run a gel?
Hello again! Thank you very much for your replies.
My protocol is the same than in conventional RT-PCR and I haven´t tried to optimize it because I only have run 2 or 3 qRT-PCR. When I started doing the conventional PCR I optimized the annealing temperature and had no changes on results.
The 7 min at the end is the final extension step and I don´t run a gel, I do the dissociation curve. I know is primer dimer looking a the dissociation curve and because the fluorescence is the same in my NTC and in my positive samples.
I am not sure what is happening. Somehow are you losing template when you do the qRT-PCR version? I do not know why primer-dimer is favored when you run the rxn that way and not favored when you do it the conventional way, if all things are equal
The only thing I can suggest, is perhaps lower your primer concentration or try a gradient such that you can manage to get rid of primer-dimer and favor your product. I hope that it will work for you.
The only thing I can suggest, is perhaps lower your primer concentration or try a gradient such that you can manage to get rid of primer-dimer and favor your product. I hope that it will work for you.
Ok!! Thank you very much! I´ll do both: try a gradient and lower my primer concentration. Tomorrow is coming a man who knows a lot about Real-Time to explain me how to use the thermocycler and optimize my reaction. I´ll try your sugestion and will post my results.
Thanks!
