Designing a Primer within an exon - Consesus required - (Nov/12/2005 )

Hello..
I have been trying to standardise QRTPCR for my gene of interest which is RAt CYP1B1.
I ran in to the following problems:
1. I used Taqman probes (spent a lot of money in doing so..). And I did a validation curve nad every good thing..only to find inconsistent results. The endogenous contro I used (18s) amplifies atleast 15 cycles before 1b1 does (even at 1/5th the concentration). This basically gives results that are exactly opposite of my SQRTPCR results. I was unsuccessful in getting the right values inspite of meddling with the threshold levels, cahnging baseline etc.
Then I found a paper that suggested the estrogen might have an effect on 18s mRNA expression

SO, now I want to use SYBR green (obviously the cheaper approach) and design my own primers.
Here is what I want to know
1. Is there any good housekeeping gene that somebody uses as their endogenous control that is known to not be affected by Estrogen (that is my treatment).
2. I tried to design exon spanning primers for 1B1 and the all the primers primer express software gave me, none of them span the 2 exons present in the gene ( also added to the fact that it is really not clear which the exons are). IS IT OK to design a primer close to the 3' end of exon 2 so that both your forward and reverse primers are in exon 2. ( if so how does that exactly work?)
3. what exactly are melting curves and how do you perform them.

I hope I can find out everything there is about it before I screw up again (it seems like that is all I have been doing fo the past few weeks!!

as to question 1, here is the best information I can give you:

http://www.ambion.com/techlib/tb/tb_151.html

perhaps pick more than one and see how they compare...they all have their pitfalls and that is the biggest flaw with real-time technology

as for 2, I do not think I can help you. the best you can do, is get RNA with no DNA contamination; make sure to run your "no RT reaction" controls and look for differences; the melting curves will tell you also if this is a problem with your primers. which leads me to question 3. which machine do you use? on the machine I use, you can set it up to run the melting curve at the end of your PCR run. this performs a melting curve analysis on your products. Based on your amplicon's Tm, you can determine if your product is pure. (you will get peaks when you run this test; one peak means one product, more peaks mean non-specific products)

I would also recommend going to ABI's website and downloading 'user bulletin #2'. there is some good information about what controls are needed and how to set them up.

1. Is there any good housekeeping gene that somebody uses as their endogenous control that is known to not be affected by Estrogen (that is my treatment).

i use cyclophyllin A. works a treat. Cyclophylin A; ACCESSION # Y00052
Forward 5’-ggcaaat gctggaccca acacaaa
Reverse 5’-ctaggcatgggagggaacaaggaa

cheers,
vetticus