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RT-PCR procedures

reaction temperature (5072C), which reduces false priming and increases the specificity of the reaction by eliminating secondary mRNA structure. Advantages of One-step RT-PCR Procedure. A one-step procedure has the following advantages:
  • Minimizes time required. The one-step reaction has fewer pipetting steps than the two-step reaction, significantly reducing the time needed to perform RT-PCR and eliminating pipetting errors.
  • Reduces the risk of contamination. The entire one-step reaction takes place in a single tube, with no transfers required and no need to open the reaction tube (steps where contamination of the PCR sample can take place).
  • Improves the sensitivity and specificity of cDNA synthesis. Two characteristics of the one-step reaction provide increased yield and efficiency: (1) the cDNA reaction is performed at a high temperature (to eliminate problems with RNA secondary structure), and (2) the entire cDNA sample is used as template for PCR.

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