HomeFAQsHow can I detect/amplify RNA targets with isothermal amplification?
FAQ: How can I detect/amplify RNA targets with isothermal amplification?
The most direct path to detecting RNA targets is to use one of our LAMP mixes optimized for detection of both RNA and DNA targets. These include the WarmStart® LAMP Kit (DNA & RNA) (NEB #E1700), which enables LAMP and RT-LAMP reactions using a variety of detection methods; and the WarmStart Colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB #M1800), which incorporates the same LAMP and RT-LAMP solution with a built-in colorimetric indicator of amplification for simple visual detection. But if alternative isothermal amplification methods are desired, then generally a combination of DNA-dependent DNA polymerase and reverse transcriptase can be used for one-step isothermal reactions. Addition of WarmStart RTx (NEB #M0380), AMV Reverse Transcriptase (NEB #M0277) or Protoscript® II MMuLV Reverse Transcriptase (NEB #M0368) is compatible with the buffer and temperature used for many isothermal amplification reactions, simplifying the reaction workflow. A separate cDNA synthesis step can be performed first, if desired, but for most detection reactions speed and simplicity are preferred. In some cases, the DNA polymerase itself can prove sufficient for single-enzyme RT isothermal amplification. Bst DNA Polymerase, Large Fragment (NEB #M0275) has been observed to have a low level of RT activity, particularly in the high magnesium concentrations of isothermal amplification reactions (~8 mM), but it is highly variable and cannot match the performance of two-enzyme mixes. Bst 2.0 DNA Polymerase (NEB #M0537)displays increased reverse transcriptase activity and can be used as an RT in some cases, but Bst 3.0 DNA Polymerase (NEB #M0374) has high reverse transcriptase activity up to 72 °C, enabling single-enzyme RT-LAMP reactions and cDNA synthesis through difficult secondary structures (see examples here: NEB #M0374). For general RT-LAMP reactions, we recommend the combination of WarmStart RTx and Bst 2.0 WarmStart (this mix is used in our LAMP Master mixes), but when a single-enzyme system is desired Bst 3.0 is the enzyme of choice.
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