Protocol for LunaScript®  RT SuperMix Kit (E3010)

  1. Mix components briefly and spin down if necessary.

  2. Prepare cDNA synthesis reaction as described below:

    COMPONENTS 20 μl REACTION  FINAL CONCENTRATION 
    LunaScript RT SuperMix (5X)
    4 µl 1X
    RNA Sample 
    variable
    (up to 1 µg)*
    Nuclease-free Water
    to 20 µl
     


    For no-RT control reaction, mix the following components:

    COMPONENTS 20 μl REACTION  FINAL CONCENTRATION 
    No-RT Control Mix (5X)
    4 µl 1X
    RNA Sample 
    variable
    (up to 1 µg)*
    Nuclease-free Water
    to 20 µl
     

    *Up to 1 µg total RNA, 1 µg mRNA or 100 ng specific RNA can be used in a 20 µl reaction. However, the cDNA input for downstream qPCR detection should typically contain < 109 copies of the target to ensure that quantitation remains linear. To accommodate larger amounts of input RNA (> 1 µg), the reaction should be scaled up to ensure optimum cDNA synthesis. 


    For no template controls, mix the following components:

    COMPONENTS 20 μl REACTION  FINAL CONCENTRATION 
    LunaScript RT SuperMix (5X)
    4 µl 1X
    Nuclease-free Water
    to 20 µl
     


    Incubate reactions in a thermocycler with the following steps:

    CYCLE STEP  TEMPERATURE  TIME  CYCLES 
    Primer Annealing
    25°C
    2 minutes
    1
    cDNA Synthesis
    55°C
    10 minutes 
    Heat Inactivation  95°C
    1 minute