This video demonstrates a quantitative method for detecting viral infection using real-time reverse transcription PCR. The reverse transcription step converts the RNA to cDNA, which is then amplified via PCR cycles. The presence of viral RNA in the sample is confirmed by analyzing the amplification and dissociation curves of DNA obtained from the PCR.
Protocol
1. Quantification of RNA Using a Micro-volume Spectrophotometer Ensure the settings of the spectrophotometer are set to RNA. Use 1-2 µL of molecular-grade water to initialize the machine and set a baseline. Use 1-2 µL of each test RNA sample to assess the RNA quantity. Save the readings and document. Adjust the RNA to 1 µg/µL, if required. NOTE: RNA must be kept on ice (or in a cool block) at all times. If RNA is obtained…
Representative Results
Table 1: Pan-lyssavirus real-time RT-PCR master mix reagents. Reagent μL/Reaction Molecular-grade water 7.55 2x Universal RT PCR reaction mix 10 Primer Forward [20 μM] 0.6 Primer Reverse [20 μM] …
Divulgaciones
The authors have nothing to disclose.
Materials
Art Barrier pipette tips (various sizes)
Thermofisher
various
Centrifuge
Beckman
Allegra 21R
Rotor capable of holding 96 well plates required. Step 3.8.