Typically, we recommend running RT-qPCR reactions as soon as possible after reactions are set up.
 
However, based on testing using purified total RNA in 96-well assays (20 ul reaction volume), Luna RT-qPCR reactions can be set up and protected from light, for up to 8 hours at 25°C and still maintain comparable performance to control reactions. Similar results were observed when testing 384-well assays (10 ul reaction volume).

In preparing 96-well assays (20 ul reaction volume) in advance with everything except sample input, we have observed similar performance when the reaction premix (step 4 in the protocol/manual) is stored for over 48 hours at 4°C (protected from light) compared to control reactions run immediately after reaction set up.