Multiplex Real-Time PCR Protocol In multiplex real-time PCR, different sets of primers with different labels are used to amplify separate genes from the template DNA in one tube. This protocol uses LUX (Light Upon eXtension) primers from invitrogen. FAM (6-carboxy-fluorescein) is used to label the gene of interest, and JOE (6-carboxy-4', 5'-dichloro-2',7'-dimethoxy-fluorescein) is used to label a housekeeping gene as an internal control to normalize between different reactions.
Real-Time PCR Protocol Protocol uses FAM-(6-carboxy-fluorescein) or JOE-(6-carboxy-4', 5' -dichloro-2',7' -dimethoxy-fluorescein) labeled LUX (Light Upon eXtension) primers, which can quantify 100 or fewer copies of the target DNA in a background of nonspecific templates, over a broad dynamic range of less than 100-107 copies. It uses uracil deglycosylase (UDG) to minimize the risk of carryover contamination, and includes a melting curve analysis of the product.
Real-Time RT-PCR: cDNA Synthesis Protocol Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA. RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue.