Q：What is the effect of RNase H activity on reverse transcription reaction?

A：

RNase H can degrade the RNA strand of the DNA/RNA hybrid. Diminished RNase H activity can avoid degradation of the RNA template of the RNA:cDNA hybrid in first-strand cDNA synthesis reaction, ensuring higher yield and synthesis of longer first-strand cDNA.

A：

The optimal reaction temperature of the standard reverse transcriptase is 42℃; but for some genes with complex secondary structures and/or high GC content, 42℃ is not enough to cause denaturing of the secondary structure, thus cDNA synthesis cannot proceed smoothly. At this point, highly thermostable transcriptase is required for cDNA synthesis, such as TransScript® II RT or TransScript®-Uni RT, which can tolerate high temperature (up to 55℃or 65℃) to ensure better results of reverse transcription. 

A：

It is necessary to assess the integrity of RNA by gel electrophoresis and ensure they are not degraded prior to reverse transcription. Do not store RNA for too long periods, and perform reverse transcription and PCR as soon as possible.

A：

When a gene is cloned from cDNA, the mutation may occur in the reverse transcription, or in the PCR process. Generally, high-fidelity Taq enzyme or Pfu enzyme are recommended for amplification, and the number of cycles should not be too high. The mutation rate can also be reduced by lowering the amount of input RNA or lowering the number of cycles. To ensure the fidelity in reverse transcription, please select a reverse transcriptase with high fidelity.

A：

For a 20 µl reverse transcription reaction, 50 ng-5μg/ 5-500 ng of Total RNA/ mRNA  are recommended. For a 50 µl PCR reaction, reverse transcription products of 2.5-5 µl (1/20 to 1/10 of the PCR reaction volume) are used as templates.