- Materials
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- Input RNA in 10 mM Tris-HCl, pH 8.0
- Consumables
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- LunaScript™ RT SuperMix Kit (NEB, cat # E3010)
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Equipment
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- Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
- Thermal cycler
- Centrifuge capable of taking 96-well plates
- Ice bucket with ice
- Optional equipment
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- PCR-Cooler (Eppendorf)
- PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
- Stepper pipette and tips
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In a clean pre-PCR hood, using a stepper pipette, or a multichannel pipette, add 4 µl of LunaScript™ RT SuperMix to a fresh 96-well plate (RT Plate).
Depending on the number of samples, fill each well per column as follows:
Plate location X24 samples X48 samples X96 samples Columns 1-3 1-6 1-12 -
To each well containing LunaScript reagent of the RT plate, add 16 µl of sample and gently mix by pipetting. If adding less than 16 µl, make up the rest to the volume with nuclease-free water.
Example for X48 samples:
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Seal the RT plate and spin down. Return the plate to ice.
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Preheat the thermal cycler to 25°C.
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Incubate the samples in the thermal cycler using the following program:
Step Temperature Time Cycles Primer annealing 25°C 2 min 1 cDNA synthesis 55°C 10 min 1 Heat inactivation 95°C 1 min 1 Hold 4°C ∞