Frequently asked questions

PCR/qPCR Kits & Reagents

• What is UNG, and how does it work?
  UNG stands for Uracil-N-glycosylase. It is an enzyme that hydrolyses uracil-glycosidic bonds in DNA containing dUTP, therefore degrading the DNA into small fragments. In this way, contamination from previous qPCR reactions can be avoided.
• What is the difference between a one-step and a two-step RT-qPCR reaction?
  In a one-step RT-qPCR reaction, the RT reaction and the qPCR reaction are done in one and the same tube. The buffer is a combination of an RT and a PCR buffer, in which both enzymes work. The one-step RT qPCR reaction is a closed tube assay, so contamination can be avoided. It saves pipetting steps and time and is easy to handle.

  
  

  In a two-step RT-qPCR, the RT reaction and the qPCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one qPCR reaction and can be archived, so that it eliminates the need to continually isolate RNA.

  
  

  Furthermore, it allows the use of oligo d(T) and random nonamer primers in the RT step and specific primers in the PCR step. This will increase the specificity and sensitivity of the assay.

  
  

• In which case is one-step RT-qPCR recommended?
  A one-step RT-qPCR is recommended when doing HTS (High Throughput Screening), where the experiment should be as easy and straightforward as possible, and when doing experiments where all sources of contamination should be excluded.
• Why is a two-step RT-qPCR kit more efficient than a one-step kit?
  In reverse transcription (RT), three types of primers can be used:

  • Oligo(dT): Binds to the RNA’s poly-A tail, transcribes only mRNA, avoids genomic DNA contamination, and yields full-length transcripts.

  • Random nonamers: Bind throughout the RNA, boosting cDNA yield by covering gaps.

  • Gene-specific primers: Bind only to the target gene, offering specificity but lower yield.Two-step RT-qPCR allows the use of oligo(dT) and random primers, maximizing both yield and transcript length. In contrast, one-step kits can only use gene-specific primers, since including random or oligo(dT) primers would risk nonspecific PCR products. At RT temperatures (40–50°C), mismatched primers may bind RNA and create off-target cDNA that gets amplified during PCR.With two-step kits, nonspecific amplification is avoided by separating RT and PCR steps—cDNA is first generated using oligo(dT)/random primers, then amplified using specific primers.