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-   -   Serotonin transporter and real time PCR (http://www.molecularstation.com/forum/real-time-pcr-quantitative-pcr-forum/86896-serotonin-transporter-real-time-pcr.html)

du_jardin 11-29-2012 02:28 PM

Serotonin transporter and real time PCR
 
Hi,

I am currently exploring changes in the serotonergic and glutamatergic system (rat brains) in respons to a treatment. I have validated primers for almost all receptors and transporters of these two neurotransmitter, except the gene for the serotonin transporter (SCL6A4). I'm using NCBI's primer blast tool to ensure specificity and netprimer to exclude primer pairs with a tendency to form primer-products (hairpins, primer-dimers and cross-dimers). I have so far tried four different primer pairs but none of them seems to produce anything near a decent amplification signal or standard curve, even though these primer pairs are specific with no primer-products and designed with the exact same guidelines in mind as all my other (functional) primers. My setup includes:

- Mx3000P
- SYBR green
- The cDNA I am using is from hippocampus and frontal cortex

Could this lack of amplification be caused by lack of SCL6A4 mRNA in these brain areas? Or does anyone have a alternative theory or experiences with this gene?

Any help is very much appreciated! :)

TV2013 02-06-2013 07:15 PM

Re: Serotonin transporter and real time PCR
 
Hi du_jardin

Did you have some answer for your question above?
I have been working with 5-HT (serotonin transporter) too and I have gotten high CT in primer 's optimization. I don┤t know how to solve this. Maybe, I could concentrating my cDNA for 1:5. My samples are from rat brain too. What do you think? Could you help me?
thanks a lot

Deborahdhw 02-11-2013 07:39 AM

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Deborahdhw 02-11-2013 07:42 AM

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Gustaveimj 02-12-2013 01:36 PM

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Elizabethsxw 02-12-2013 01:37 PM

エアジョーダン 2013
 
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Gustaveimj 02-12-2013 01:48 PM

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Gustaveimj 02-12-2013 02:31 PM

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Elizabethsxw 02-12-2013 03:49 PM

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