SYBR and TaqMan - (Feb/13/2009 )

I am an undergrad doing some RNA isolation then gene expression analysis through PCR.

I am using real time PCR. Although I have used it several times I am still a novice at it.

I have been using SYBR green but now switched over to TaqMan due to a amplification kit I had ordered due to the extreamly low amounts of RNA I was able to isolate.

I know the mechanism behind TaqMan, but will the SYBR primers work for for TaqMan?

Has anyone used an amplification kit before? No one else in my lab has so I am learning by trial by fire.

Thanks,

Tagman requires a probe besides the two primers. The three things usually come together from the vendor such as ABI. You should not not use SYBR primers in Taqman PCR if you don't have compatible probe.

Many studies including my own testing have shown the these two methods give comparable results.

It really depends. First thing to check is if the primers are compatible with the probe you are using (eg: probe does not overlap w/ primer targets, and there is no major cross reactions). We use a combination of Primer Express and Primer Premier 4 for these purposes, but there are other software packages out there that should do the same trick.

Also, make sure that your cycling conditions match your probe (ie: remove the melt curve step, and make sure that there is sufficient Tm difference between your primers and probe - the probe should be ~ 10C higher, usually around 68 or so)

In our experience, SYBR tends to give earlier crossing points, but this is frequently due to non-specific signals from non-specific amplification or primer dimer. Taqman (or for that matter any probe system) will give you that extra level of specificity, and that's why we soley use Taqman probes as the basis for our quant assays.

We use a lot of PCR kits, and they have a lot of advantages, but really depends on your needs. It can sometimes be cheaper, and definitely much more conveniant. They also take out the variability component, if you need to evaluate and maintain your assay over long periods of time (this is esp. important in diagnostics, but also in quant work). A drawback is that a lot of the all-in-one kits already have pre-mixed magnesium, so it can be difficult to adjust your PCR if you need less of the metal.

Try using the primer3 software.
You can insert your sequence and the primers you've been using and then hit the "pick primers" button.
It might be able to find a probe that will work with it.
It's worth a try.

pcrman on Feb 14 2009, 03:25 AM said:

Hi pcrman,

we just started working in lab which has the ABI one step plus real-time instrument. is it possible to order primers separately and use the master mix and the cDNA synthesis kit for other companies, or is it advisable to order everything from ABI?

Thanks

Pmaj

ellipis05 on Feb 13 2009, 12:03 PM said:

Hi,
SYBR Green primer pairs can work for Taqman assays. Try using Beacon Designer from Premier Biosoft - http://www.premierbiosoft.com/molecular_beacons/index.html
In the TaqMan mode, you can import SYBR Green primers and design a compatible probe. You can analyze your primer and probes using the Free Edition at: http://www.premierbiosoft.com/qpcr/index.html

Wilson

I didn't want to start a new thread. I had a question about Beacon Designer 7. I have designed a taq man and SYBR green primer set for a certain gene, but none of the primer designs span an intronic region. How do I force or design a primer to include on intron sing this program?