1. Measure RNA via NanoDrop to determine volume of 1 ug.

2. cDNA Synthesis:

• Thaw template RNA on ice

• In PCR tubes:

• Prepare 16 ul RNA reaction [1 ug].

• Add 4 ul of iScript RT supermix (BioRad 1708841) to 16 ul RNA reaction (total ~20 ul)

• RT-PCR reaction protocol:

• Priming: 5 min at 25°C

• Reverse transcription: 30 min at 42°C

• RT inactivation: 5 min at 85°C

• Store cDNA at -20°C or proceed with quantitative real-time PCR (qPCR).

• Ideally no-RT control supermix should be used to check for genomic DNA contamination

3. qPCR with SensiFast SYBR Kit

• Sign up to reserve Roche Real Time Cycler 1 via microsoft outlook.

• Switch on lightcycler to preheat (room A114).

• If computer needs restart, Login: lcinstall, Pass: LCinstall

• Start lightcycler480 software (Login: Kapahi, Pass: Roche480)

• denature: 1: None

• PCR cycles: 40: Quantification

• melt: 1: Melting Curves

• cool: 1: None

• Target: 95; Acquisition Mode: None; Hold: 00:02:00; Ramp Rate: 4.8; Sec Target: 0; Step Size: 0; Step Delay: 0

• Target: 95; Acquisition Mode: None; Hold: 00:00:05; Ramp Rate: 4.8; Sec Target: 0; Step Size: 0; Step Delay: 0

• Target: 60; Acquisition Mode: Single; Hold: 00:00:20; Ramp Rate: 2.5; Sec Target: 0; Step Size: 0; Step Delay: 0

• Target: 95; Acquisition Mode: None; Hold: 00:01:00; Ramp Rate: 4.8

• Target: 40; Acquisition Mode: None; Hold: 00:01:00; Ramp Rate: 2.5

• Target: 65; Acquisition Mode: None; Hold: 00:00:01; Ramp Rate: 1

• Target: 95; Acquisition Mode: Continusous; Ramp Rate: 0.02; Aquisitions: 25

• Target: 40; Acquisition Mode: None; Hold: 00:00:10; Ramp Rate: 2.5; Sec Target: 0; Step Size: 0; Step Delay: 0

• create new experiment from template (SensiFast SYBR)

• Save Experiment (floppy disk symbol)

• Use 4titude white plate (BASIC Scientific 4ti-0381) on cooling plate on ice.

• Dilute primer pairs to 5 uM per single primre (1:20, if stock 100 uM) in RNase-free water. 

• Final concentration in qPCR reaction: 500 nM

• Dilute cDNA reaction with RNase-free water to have sufficient amounts

• 20 ul cDNA + 180 ul water = 1:10

• dilution can be much higher (1:50); amount of cycles to reach c_p value is increased

• Add 3 ul cDNA to 4titude plate.

• Prepare master mix for each primer pair

• 5 ul SYBR-Mix

• 2 ul Primer pair

• 3 ul diluted cDNA

• = 10 ul q PCR reaction

• Add 7 ul of master mix to appropriate wells. Reaction volume per well is 10 ul.

• Close plate with adhesive film (VWR 60941-078)

• Spin plate shortly

• Insert plate into lightcycler and start run (~55min)

4. Analyze qPCR with Lightcycler software

• Press analyze button (method: Abs Quant/2^nd derivative)

• Press calculate button after selecting the right wells

• In sample window: right click to export data table

• In amplification window: right click to export chart as jpeg

• Press analyze button (method: T_m calling)

• In melting peaks window: right click to export chart as jpeg

• Save data and close application