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qRT-PCR with SensiFast SYBR Kit (BioLine BIO-98020)
Author: Jennifer Beck, updated date: , view: 6, Q&A: 0
Tags: RNA and qRT-PCR

Procedure

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 cp 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/2nd 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: Tm calling)

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

  • Save data and close application


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