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Designing a Successful qRT-PCR Experiment


A Successful One Step qRT-PCR Experiment Requires Careful Consideration of Several Important Factors:

Detection Method: To determine if your gene of interest is represented in an RNA sample, end point PCR followed by gel based staining is suitable. For target quantitation, real-time PCR with SYBR Green I or TaqMan probes are recommended. Many researchers find TaqMan probes to be the most sensitive real-time PCR detection platform. Ambion's MessageSensor RT Kit is compatible with all of these methods.

Designing Primers and Probes: We recommend using primer design software for both TaqMan and SYBR Green I applications to improve your assay design. The software program should accept defining parameters such as melting temperature (Tm) and reaction conditions. Choose the TaqMan probe sequence prior to the primers since probe design is key for successful detection (there are some programs that will choose probe and primer sequences simultaneously such as Primer Express (ABI)). For eukaryotic targets, use primers that overlay an intron-exon junction to eliminate signal from genomic DNA contamination. For best results, amplicon lengths of 80 to 200 bp are recommended.

Primer and Probe Concentrations vs. Target Abundance: Gene-specific primer and TaqMan probe concentrations should be
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