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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