For two-step RT-PCR, random hexamers, oligo (dT)16, and sequence-specific reverse primers can all be used. The choice of primers for reverse transcription is best made after experimentally evaluating all three priming systems.
Life Technologies has developed a comprehensive set of guidelines to ensure success when using real-time PCR reagents and instrumentation. These guidelines are simple and easy to follow. Many traditional variables, such as magnesium chloride concentration and the thermal cycling protocol itself, have been standardized, greatly reducing assay development time.
All Life Technologies Real-Time PCR reagent kits contain a passive internal reference to normalize non-PCR-related fluorescence fluctuations. Normalizing with a passive internal reference minimizes well-to-well variability that can result from a variety of causes, such as pipetting errors and sample evaporation. The passive reference is also essential for accurate results when multiple probes (with different reporter dyes) are combined in a single tube.