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Dealing with Reverse Transcription and Amplification Inhibitors: Reagent Choice Matters

Rod joined the Promega Genetic Analysis team in 2011 as a Senior Research Scientist.  In this role, he is responsible for leading a team of R&D scientists in the development of amplification technologies and products. Before joining Promega, Rod was a plant molecular virologist/biologist and completed his Ph.D. at Oklahoma State University (OSU) prior to post-doctoral stints at OSU, the University of Kentucky, and with the USDA-Agricultural Research Service in Temple, TX.  From 1998-2010 he worked at Roche Diagnostics in Indianapolis where he filled a number of roles, most of which focused on reagents and instrumentation for nucleic acid purification and real time PCR.
  • Rod Pennington, Ph.D.

  • Senior Research Scientist

  • Original Webinar Date: Tuesday, February 11, 2014

Depending on the nucleic acid purification method, purified nucleic acids may contain any number of compounds with the potential to affect downstream reactions such as reverse transcription or PCR amplification. With the goal of helping you improve these enzymatic reactions, we will discuss the various types and sources of potential inhibitors and their potential impacts including data contrasting the performance of different downstream reagents in the presence of inhibitors. Learn additional information below.

- Additional Information

Whether performed separately or together in one reaction, reverse transcription (RT) and amplification by PCR are enzymatic processes and consequently are subject to inhibition by a variety of compounds.  RT and PCR inhibitors exert their effects through direct interaction with the RNA or DNA template, the polymerase itself, or by interacting with other critical reaction components such as enzyme cofactors.  A common source of RT and PCR inhibitors is the sample material from which the nucleic acid template is isolated.  Ideally, RT and PCR inhibitors are removed during sample prep, but it is still common for inhibitors to survive the nucleic isolation process.

Depending on the enzyme and the buffer formulations, there are often large differences between RT and PCR reagents in terms of their resistance to inhibitors, which are often impossible to completely avoid in the purified nucleic acid samples. As such, the choice of RT and PCR reagents is an important factor influencing the outcome of these enzymatic reactions and hence, the downstream results.  This webinar will discuss the types of RT and PCR inhibitors, their sources and possible modes of action. Experimental methods to detect and characterize inhibitors will also be discussed. Finally, we’ll present data demonstrating the performance of RT and amplification enzymes in the presence of inhibitors in order to help guide your enzyme choice and improve the success rate of your experiments.