To begin, prewarm a thermal incubator at 42 degrees Celsius. Add one microliter of magnesium acetate and potassium acetate solution mix to the side of the tube containing the lyophilized, pre-mixed RTRPA pellet. Resuspend the pellet in 12.4 microliters of the RNA sample.
To initiate the RTRPA reaction, briefly spin down the added solution using a spin down mini centrifuge at room temperature for three seconds. Carefully tap the tube to mix the contents thoroughly and spin down again. Incubate the reaction at 42 degrees Celsius for 60 minutes in a preheated thermal shaking incubator.
After the reaction has ended, remove the reaction tubes and place them on ice before proceeding to the CRISPR-Cas detection. For nucleic acid detection, turn on a fluorescence microplate reader. Set and preheat the microplate reader to 37 degrees Celsius and select the program.
Then place a 384 well plate on ice. To resuspend the lyophilized CRISPR-Cas pre-mixed detection reaction, add 17 microliters of eight millimolar magnesium chloride solution. Mix by carefully tapping the tube.
Briefly spin down for three seconds before placing the tube on ice. Transfer 18 microliters of the resuspended reaction mix to each well of the pre-cooled 384 well plate on ice. Add two microliters of the RPA product prepared to each reaction well.
Remove the plate from the ice. Quickly place it into a preheated fluorescence microplate reader and press Start. For lyophilized, pre-mixed Cas13a-based reactions, either 6%trehalose or 2%sucrose preserves the reaction efficiency well after lyophilization.
The lyophilized pre-mixed reagents for RTRPA and Cas13-based reactions are stable at 20 degrees Celsius for at least eight months.
