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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 adjusted according to target abundance. By performing a primer titration, an optimal final primer concentration for a given target can be found. This can vary between 150 to 500 nM. An initial concentration of 400 nM for each primer is a useful starting point. The optimal TaqMan probe concentration can also vary according to relative target abundance, though 80 nM is generally a good starting point. We recommend a titration of 50 to 800 nM probe for optimization.

RNA Sample: The quality of the starting RNA template plays an important role in RT-PCR results. Ambion recommends RNAqueous-4PCR to obtain RNA free of DNA. An RNA sample containing contaminating DNA can be treated with Ambion's DNA-free Reagents to help eliminate genomic DNA contamination. To synthesize large amounts of RNA for a standard curve, Ambion recommends the MEGAscript High Yield Transcription Kit.

Control Reactions: Always include at least two controls in your experiments. The first controls for genomic DNA contamination by excluding RT from the reaction (minus RT control). The second, which contains all master mix components except the input RNA, controls for contamination of reagents and the reaction tubes (no-template control). Any other deviations from the master mix will also need appropriate controls.

Master Mix Preparation: A potential source of DNA contamination often arises from prior PCR runs. It is highly recommended that the pre-PCR setup occur at a separate location from post-PCR sample analysis, and that separate pipettes, tips and reaction tubes be used. RT-PCR is highly sensitive to small changes created by pipeting differences and airborne substances. Additionally, Ambion's DNAZap DNA Degradation Solution can be used to decontaminate thermal cyclers, pipettes, and other surfaces prior to RT-PCR.

For a helpful general resource for qRT-PCR approaches and strategies, Ambion recommends the following references: Bustin SA (2000) J Mol Endocrinol 25: 169-93 and Bustin SA (2002) J Mol Endocrinol 29: 23-39.


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