- Materials
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- Input influenza RNA
- Influenza A primers
- Influenza B primers
- Consumables
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- SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (ThermoFisher, cat # 12574018 or 12574026)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with heat seals
- 1.5 ml Eppendorf DNA LoBind tubes
- 5 ml Eppendorf DNA LoBind tubes
- 15 ml Eppendorf DNA LoBind tubes
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Thermal cycler
- Qubit fluorometer (or equivalent for QC check)
- Multichannel pipettes suitable for dispensing 20–200 μl, and tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Magnetic rack suitable for 96 well plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher CAT#12027)
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Microfuge
- Optional equipment
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- PCR hoods with UV steriliser
- PCR-Cooler (Eppendorf)
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To reduce risk of contamination, we recommend the use of PCR hoods with a UV steriliser when setting up the PCR plates.
- When handling the primer stocks and derivatives, use a clean template-free PCR hood.
- When handling the samples and/or a positive control, use a clean template-addition PCR hood.
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In a clean template-free pre-PCR hood, prepare the primer mixes for influenza A and influenza B as follows in 1.5 ml Eppendorf DNA LoBind tubes:
Note: The volume requirements can be adjusted according to stock concentrations and experiment needs.
Influenza A primer mix
Primer Concentration Volume Nuclease-free water - 378 µl Tuni 12 100 µM 16.8 µl Tuni 12.4 100 µM 4.2 µl Tuni 13 100 µM 21 µl Total 420 µl Influenza B primer mix
Primer Concentration Volume Nuclease-free water - 378 µl B-PBs-UniF 100 µM 5 µl B-PBs-UniR 100 µM 5 µl B-PA-UniF 100 µM 2.5 µl B-PA-UniR 100 µM 2.5 µl B-HANA-UniF 100 µM 5 µl B-HANA-UniR 100 µM 5 µl B-NP-UniF 100 µM 3 µl B-NP-UniR 100 µM 3 µl B-M-Uni3F 100 µM 1.5 µl B-Mg-Uni3F 100 µM 1.5 µl B-M-Uni3R 100 µM 3 µl B-NS-Uni3F 100 µM 2.5 µl B-NS-Uni3R 100 µM 2.5 µl Total 420 µl -
In the template-free pre-PCR hood, prepare the following master mixes in Eppendorf DNA LoBind tubes and mix thoroughly as follows:
For X12 samples, use 1.5 ml Eppendorf DNA LoBind tubes:
Component Influenza A RT-PCR Master Mix Influenza B RT-PCR Master Mix Nuclease free water 280 µl 280 µl Influenza A primer mix 28 µl - Influenza B primer mix - 28 µl 2X Reaction Mix 350 µl 350 µl SuperScript™ III RT/Platinum™ Taq Mix 28 µl 28 µl Total volume 686 µl 686 µl For X24 samples, use 1.5 ml Eppendorf DNA LoBind tubes:
Component Influenza A RT-PCR Master Mix Influenza B RT-PCR Master Mix Nuclease free water 560 µl 560 µl Influenza A primer mix 56 µl - Influenza B primer mix - 56 µl 2X Reaction Mix 700 µl 700 µl SuperScript™ III RT/Platinum™ Taq Mix 56 µl 56 µl Total volume 1372 µl 1372 µl For X48 samples, use 5 ml Eppendorf DNA LoBind tubes:
Component Influenza A RT-PCR Master Mix Influenza B RT-PCR Master Mix Nuclease free water 1120 µl 1120 µl Influenza A primer mix 112 µl - Influenza B primer mix - 112 µl 2X Reaction Mix 1400 µl 1400 µl SuperScript™ III RT/Platinum™ Taq Mix 112 µl 112 µl Total volume 2744 µl 2744 µl For X96 samples, use 15 ml Eppendorf DNA LoBind tubes:
Component Influenza A RT-PCR Master Mix Influenza B RT-PCR Master Mix Nuclease free water 2240 µl 2240 µl Influenza A primer mix 224 µl - Influenza B primer mix - 224 µl 2X Reaction Mix 2800 µl 2800 µl SuperScript™ III RT/Platinum™ Taq Mix 224 µl 224 µl Total volume 5488 µl 5488 µl -
For each influenza type, place one clean 96-well RT-PCR plate into a PCR-cooler (if using).
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Using a stepper pipette or a multichannel pipette, aliquot 49 µl of influenza A RT-PCR Master Mix into the influenza A RT-PCR plate.
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Using a stepper pipette or a multichannel pipette, aliquot 49 µl of influenza B RT-PCR Master Mix into the influenza B RT-PCR plate.
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Seal the RT-PCR plate(s) and transfer to a template-addition pre-PCR hood.
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Transfer 1 µl of influenza A samples to the wells containing influenza A RT-PCR Master Mix in the influenza A RT-PCR plate and mix thoroughly by pipetting the contents of each well up and down.
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Transfer 1 µl of influenza B samples to the wells containing influenza B RT-PCR Master Mix in the influenza B RT-PCR plate and mix thoroughly by pipetting the contents of each well up and down.
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Seal the RT-PCR plate(s) and spin down in a centrifuge.
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Incubate the influenza A RT-PCR plate using the following program, with the heated lid set to 105°C:
Step Temperature Time Cycles cDNA synthesis 42°C 60 min 1 Initial denaturation 94°C 2 min 1 Denaturation
Annealing and extension94°C
45°C
68°C30 sec
30 sec
3 min
5Denaturation
Annealing and extension94°C
57°C
68°C30 sec
30 sec
3 min
31Hold 4°C ∞ -
Incubate the influenza B RT-PCR plate using the following program, with the heated lid set to 105°C:
Step Temperature Time Cycles cDNA synthesis 45°C 60 min 1 cDNA synthesis 55°C 30 min 1 Initial denaturation 94°C 2 min 1 Denaturation
Annealing and extension94°C
40°C
68°C20 sec
30 sec
3 min 30 sec
5Denaturation
Annealing and extension94°C
58°C
68°C20 sec
30 sec
3 min 30 sec
30Final extension 68°C 10 min 1 Hold 4°C ∞ -
Optional actionIf necessary, the protocol can be paused at this point. The samples should be kept at 4°C and can be stored overnight.
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Resuspend the AMPure XP beads by vortexing.
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Add 50 µl of resuspended AMPure XP beads to each well of the RT-PCR plate(s) and mix by gently pipetting.
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Incubate the PT-PCR plate(s) at room temperature for 10 minutes.
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Prepare at least 500 µl 80% ethanol in nuclease-free water per sample.
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Spin down the RT-PCR plate(s) and pellet the beads on a magnet for 5 minutes. Keep the plate on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
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Keep the plate on the magnet and wash the beads in each well with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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Spin down and place the plate back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
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Remove the plate from the magnetic rack and resuspend each pellet in 15 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 15 µl of eluate containing the DNA per well, into a clean 96-well plate(s).
Dispose of the pelleted beads.
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Quantify 1 µl of each eluted sample using a Qubit fluorometer.