RNA extraction, RT and cDNA preparation, qPCR Protocol
RNA Extraction
  • RNA extraction is performed using the Qiagen RNeasy Mini Kit (cat 74104). DNase I is prepared and aliquoted for use as described in the kit manual. Buffer RDD is stored at 4ºC.
  • RNA concentration should be measured using a Nanodrop or Qubit immediately after extraction and stored on ice for immediate use or transferred to -80ºC if not used immediately.
RT and cDNA Preparation
  • We use Superscript VILO MasterMix (Invitrogen cat# 11755) for cDNA preparation
  • If preparing cDNA for qPCR normalize RT substrate content to the sample of lowest RNA yield to ensure comparable cDNA concentrations across samples for qPCR.
  • Prepare the 10µl reaction mixture below on ice in 200µl PCR tubes for each sample. (This reaction may be scaled up to 100µl).
    ComponentVolume (µl)
    SuperScript VILO Master mix 2
    RNA (up to 1.25µg) X
    DEPC-treated or nuclease-free water up to 10
    • Gently mix and incubate in a thermal cycler as below:
      TemperatureTime
      25ºC 10 minutes
      42ºC 60 minutes (can be doubled to increase yield)
      Hold at 85ºC 5 minutes
      • cDNA should be diluted 10x (add 90µl of nuclease-free dH2O to each sample)
      • Store cDNA on ice until ready for use or at -20ºC long term.
qPCR:
  • We use 2x SYBR Green Master mix (Thermofisher cat# 4309155)
  • Prepare the 25µl reaction mixture below for each sample:
    ComponentVolume (µl)
    2x SYBR Green Master Mix 12
    cDNA (diluted or undiluted) 1
    Nuclease-free water 11
    10µM F+R primer mix 1
    • To reduce pipetting error cDNA and primers can be prepared in nuclease free water and dispensed into the wells of a 96-well qPCR plate. 2x SYBR Green master mix can then be added across all wells using a multi-channel or multi-dispensing pipet.
    • The plate should be kept on ice, sealed and centrifuged before loading into qPCR machine.
    • We use a Biorad CFX 96 Touch. The experiment below should be set up using the software:
      CycleStepTemperatureTime
      Initial Activation 95ºC 15 minutes
      40 cycles Denaturation 94ºC 15 seconds
      Annealing 60ºC 30 seconds
      Extension 72ºC 30 seconds
      Data AcquisitionTemperature
      Melt Curve 65ºC 0.5ºC increment
      95ºC 0.5ºC increment