Trans RT-PCR series consists of five types of reverse transcriptases without RNaseH activity, avoiding the degradation of RNA template during the first- strand cDNA synthesis, thus guaranteeing the yield and length of the first-strand cDNA synthesis.
Every Trans reverse transcriptase has a corresponding first-strand cDNA synthesis SuperMix, as well as the corresponding two-step and one-step RT-PCR SuperMix. For first-strand cDNA synthesis SuperMix, in addition to RNA templates, primers and RNase-free Water, only 2 x Reaction Mix and RT/RI Enzyme Mix need to be added without thermal denaturation and ice bath, making procedures simplified and contamination reduced.
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.
Q: What is the difference between the reverse transcriptions at different temperatures?
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.
Q: There is no product after RT-PCR?
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.
Q: Why do mutations often occur after gene cloning and sequencing?
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.
Q: How much template is typically used for RT-PCR?
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.