Chapter 2 Designing an RQ Experiment
Selecting One- or Two-Step RT-PCR
12 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
Selecting One- or Two-Step RT-PCR
When performing Real-Time PCR, you have the option of performing reverse
transcription (RT) and PCR in a single reaction (one-step) or in separate reactions (two-
step). The reagent configuration you use depends on whether you are performing one-
step or two-step RT-PCR:
• Two-step RT-PCR is performed in two separate reactions: first, total RNA is reverse
transcribed into cDNA, then the cDNA is amplified by PCR. This method is useful
for detecting multiple transcripts from a single cDNA template or for storing cDNA
aliquots for later use. AmpErase
®
UNG (uracil-N-glycosylase) enzyme can be used
to prevent carryover contamination.
IMPORTANT! This guide assumes that RQ experiments are designed using two-
step RT-PCR. For additional options, refer to the Real-Time PCR Systems
Chemistry Guide.
IMPORTANT! RQ plates may be run with either standard or Fast thermal cycling
conditions. An RQ study must be composed of RQ plates of the same thermal
cycling protocol. RQ plates run on standard thermal cycling protocols and RQ
plates run on Fast thermal cycling protocols cannot be combined into a single RQ
study.
• In one-step RT-PCR, RT and PCR take place in one buffer system, which provides
the convenience of a single-tube preparation for RT and PCR amplification.
However, you cannot use the carryover prevention enzyme, AmpErase
®
UNG, with
one-step RT-PCR. For more information about UNG, refer to the Real-Time PCR
Systems Chemistry Guide.
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