designing QRT-PCR Primers - (Nov/07/2005 )
Really need some advice here.
I'm working on transcriptional pausing in a gene (yay for me!).... anyway, the pausing site occures within intron 1 of the gene. i've been asked to design some qrtprimers to do some real time... to show that the pausing is removed after treatment with hormones. i said, sure, ok.....oh wait.
so, a couple of questions:
is it possible to use primers that are kept within exon 1... instead of crossing over to exon 2?
what i'm thinking is, have two sets of primers... one that'll be within the first exon and another set , say, exon 2/3. run the cDNA that was no treated.... look at the difference between the two different products....
or..... design one set of primers that cross exon1/2. run the cDNA, and look if there's an increase in the product made?
are there any good qrt-pcr designing programs that are free?
does sybr green require anything special?
the reason to design primers that span introns is to lessen the likelihood of gDNA contaminating your reaction, so that if you get gDNA in your RNA prep hopefully it won't screw up your product.
you don't have to use this technique, just be sure to do all your controls (RNA template with no RT reaction being the relevant control if you don't use intron-spanning primers) to make sure your product is specific; and for gosh sakes whatever you do, run melting curves
I think either strategy would work, although the second looks a bit easier. just make sure to use a good RNA prep method that gets rid of DNA (some actually add DNAse to their preps; I use Stratagene's Absolutely RNA kit without adding the DNAse and I've never had a problem, after almost two years of running qPCR's)..the important thing is to find a method that gives you good clean RNA
and did I mention, run your melting curves? if you use SYBR green this is essential. Invitrogen's website has a free primer design function, but you still have to manually check for dimers and hairpins, I don't care what they tell you...someone else may know of a better free software?
Um...if all you have to do to satisfy your demanding boss is to show increase in expression after hormone treatment, you can use intron-spanning primers, I think. all you are looking for is increase in gene product, correct?
you know what, this has got be completely baffled.
i've already run several real times, shown that there is an increase in teh gene product. i think this next 'investigation' is supposed to be a bit more 'specific'.
my RNA is squeaky clean. yay. but in any case, hmmmmmm, no good primers located just in exon 1. the best i could come up with spans exon 1/2....
is there a way to look at the RNA before the introns get chopped out of it?
thaks a million,
Although I'm pretty competent with real-time, I'm ignorant as far as studying transcriptional pause sites, so please excuse me if this is lame...
would it not be most relevant to design something like ChIP, for the mRNA just after transcription? Is this possible? I think what you are really looking for is binding state of the first exon's pause site? how can real-time PCR show you this, and give you the information you need? I am thinking it would almost have to take some sort of pulldown assay for binding state, but I don't know how you would do it.
the only way i thought of was to use a nuclear run on... which is already in the hat... so i'm a little cough*very*cough confused as to why real time should be used.
well.... the more i look at my list of things to do.... this one just doesn't make much sense. maybe i'll let it slide and see if anyone notices.
anyway, thanks a bunch!
just in case somebody will notice and you ll have to do it after all - or someone needs answers to your initial questions:
- you can use Ensembl to display the full exon/intron sequence, like HERE .
- a free RTPCR-Primer design tool is AutoPrime, which fetches sequence from Ensembl and uses Primer3 to find primers either on exon borders or within different exons, that are far apart.
Good luck anyway,
found out more info..... and getting a protocol for it.
we're going to be looking at nuclear RNA, before it becomes mRNA. mmm low abundance RNA.
i get my sequence, use the free oligoperfect online design tool on the invitrogen site [75-250 bp, Tm optimum 62, 22bp length optimum etc etc], take primers they suggest from there and type them into the free netprimer online tool and choose the best pair [after blasting of course].... the primers work perfectly 8/10 times
Have you noticed that these two programs give a very different melting T for the same primers?
yes they do, and they are usually lower than the Tm which comes with the primers when they are produced!