It’s possible to use RNA template with a TwistAmp® kit just by adding a suitable reverse transcriptase (RT) when setting up a reaction. The only difference between a TwistAmp® exo, and a TwistAmp® exo RT kit is the addition of an M-MLV RT to the freeze dried reaction. If an RT that works at 37–42 °C is added to RPA chemistry then RNA can be reverse transcribed and the cDNA produced and amplified all in one step. The complementary DNA sequence will be that of the RNA added. We recommend using similar RT amounts to that of a PCR reaction of the same volume, according to your chosen manufacturer’s instructions. We also suggest that you run reactions at 40°C and delay agitation of the reaction by a minute to give the RT time to work, otherwise you just run the reaction as you would a normal TwistAmp® reaction (it’s a one-step process). It’s also advisable to add RNase Inhibitor to any reaction where RNA is the target material.
T8 and T16 devices are still available by contacting Axxin directly at firstname.lastname@example.org or www.axxin.com. For end point detection methods any incubator that can maintain a constant temperature between 37-42°C can be used. RPA real-time fluorescence detection can be carried out on various available incubating fluorometers or alternatively any plate reader or real-time thermal cycler that can excite and detect the chosen fluorophores and hold a steady temperature of 37-42°C. The reaction should be transferred into an appropriate reaction vessel if the device does not fit 0.2ml tubes (e.g. a multi-well plate), and incubated/monitored according to the requirements of the device. Furthermore, the agitation regime should be adapted to mimic the protocol. The frequency of fluorescence reading of the reactions can be determined by the user (commonly every 20-30 seconds). When using a thermocycler, you may need to change the supplied reaction tube caps from the domed ones provided. Many fluorometers either read from the base up, or through the side. If however, your thermocycler reads fluorescence from above the tube, flat lids may be required. Thermocyclers usually have heated lids (it’s normally a set temperature that users can’t adjust), which on some models can be switched off. The temperature they are normally set to is too high for RPA, and may affect the efficiency of the enzymes. Heated lids are used to prevent PCR reactions evaporating and condensing on the lid, however, RPA is run quickly, and at a low temperature, so that this isn’t an issue. We recommend switching off the heated lids where possible.
Both TwistAmp® exo and fpg kits can be used for measuring amplicon in real-time by reading fluorescent probe signal. Generally speaking the kinetics of probe cutting and therefore fluorescence accumulation tends to be faster for TwistAmp® exo. If you are interested in measuring end point amplification products then we recommend using a TwistAmp® nfo kit in combination with an exo probe as the nfo nuclease will successfully cut the probe, but not reduce the final overall yield of amplified material in contrast to the use of the TwistAmp® exo kit where it is not possible to measure end point exo reactions due to the exonuclease present in the reaction mixture digesting most of the amplified product once amplification has ceased.
TwistDx is continuing to sell separately TwistAmp® exo positive control template and oligonucleotides, to provide the customer with a ready-made assay to test with.
If you have any questions, or require any further information please don’t hesitate to contact our customers support team at email@example.com