multiplex RT-PCR - (Jan/29/2007 )

hi
? have anyone heard about omething called multiplex RT- PCR ?

IM ASKING BECAUSE in my lab we dont do carrier detection of Duchene carriers females coz we dont have southern blot but we heared that another lab performs multiplex RT- PCR for female carriers ???
the problemm is that this order is x linked and so a female would have 2 copies so we need gene dosage to detect the carrier is that possible by THAT SO CALLED MULTIPLEX RT PCR?
or is it bettter done with real time PCR or southern blot?

---

Multiplex PCR is a 'normal' PCR but it enable simultaneous amplification of many targets of interest at the same time in one single reaction by using more than one pair of primers in the same reaction tube. If genotyping Duchenne implies detection of more multiple markers, multiplex is very useful. If not, i think a typical PCR is enough.

---

hi

thanks for ur reply

---

Aleruiz,
Do you know if you need any sort of internal control with the multiplex? I guess I am asking if a multiplex can be used for quantification.
Thanks!

---

Yes, you need the internal control for quantification. You can amplify a known fragment of a housekeeping gene or use rRNA from your samples to normalize. Just remember be so carefully when design all primers to be used in the multiplex, your amplified fragments should be very specific and reliable.

---

OK - thanks!
mito 1

---

One problem encountered when multiplexing is if one target is present in a much higher copy number than another, it can outcompete for components of the reaction and affect the efficency of the second reaction. Sometimes dropping the primer concentration of the higher copy number target can help reduce this competition.

---