Basically, there are three kinds of real-time PCR (qPCR):

ONE-STEP REAL-TIME PCR

• Use purified RNA.
• The two reactions of cDNA synthesis by RT (reverse transcription) and real-time PCR happen in one tube.
• Fewer steps reduce possible contamination.
• Less pipetting ensures more accurate results.
• Useful for high-throughput qPCR

TWO-STEP REAL-TIME PCR

• Use purified RNA.
• Two separate reactions, cDNA synthesis and real-time PCR, in separate tubes.
• More efficient because random or oligo dT primers can be used.
• Offers possibility to store cDNA for future use.
• Can use one cDNA sample for multiple assays with different primers

DIRECT REAL-TIME PCR

• Use purified RNA, or use sample directly, i.e., no purification of RNA is needed.
• Use sample directly, i.e., no purification of RNA is needed.
• Reaction system optimized for using cell lysate directly for cDNA synthesis and real-time PCR
• Cell lysate can be stored for future use.
• Reduces errors and contamination.
• Best for rare or unique samples and high-throughput screening.

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