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Reverse transcriptase PCR

Reverse transcriptase PCR - Molecular Biology Techniques

Reverse transcriptase PCR - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 04-27-2010, 07:34 PM
Pipette Filler
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Default Reverse transcriptase PCR



Dear All
I have the pleasure to ask about?
I am going to extract mRNA and produce cDNA of specific gene
what is the Ideal methodology to produce it
the cycles of PCR how can I do it for the best annealing of the primer and cDNA amplification,
3- I just want to detect that a gene was expressed or now, not qPCR.
4- i have to use B actin or what?
hoping to get all information and if any one has detailed explantion i will be greatfully happy.
please.
Hani
if any oen has something should you send it through e mail
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Old 05-11-2010, 04:32 PM
Pipette Filler
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Default Re: Reverse transcriptase PCR

To make your cDNA you can use 1 or 2 step RT-qPCR but this will add a fluorescent dye which you say you don't want because this allows real-time quantisation. If you can find a RT-PCR reagent without SYBR Green you could do it qualitatively like you want. Reason im saying this; i'm not sure if SYBR Green is going to compete with Ethidium Bromide as a dye for A/PAGE. Remember that they are both interclating type dyes that bind to dsDNA and idt that SYBR can be picked up by UV excitation like in agarose/PAGE. That way you can transfer your RT-PCR amplicons to an agarose/polyacrylamide gel for electrophrosis to confirm presence/absence of your target.

B-actin is a reference gene (or housekeeping) that is used to normalize expression between different groups i.e cancer tissue vs normal tissue. QiAgen has a good writing about this, too detailed for me to write here. For more info read the the post "WHAT and WHY Normalization?" post in this forum.


I suggest you read

"Real Time PCR Applications Guide" from biorad

&

"Critical Factors for Successful Real-Time PCR - English (PDF)" from

qiagen.com/literature/brochures/category.aspx?ID=304 <<<great guide

Last edited by muffinman563; 05-12-2010 at 10:25 AM.
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