HiroshiMaeda:qPCR

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qPCR analysis using Applied Biosystems StepOnePlus™ Real-Time PCR System

HiroshiMaeda Protocol: 2011.2.20 last updated


<Design Primers using PrimerExpress>

  • select TaqMan design
  • change parameters of PrimerExpress
    • amplicon length: 100 bp
    • primer length: from 15 bp
    • show results: 200
  • design closer to 3’end if possible (less sensitive to 5’RNA degradation, cDNA synthesis efficiency)
  • design F/R primers on two different introns if genomic seq is available.


<RNA isolation>

  • Harvest at least 3 biological replicates (each replicate have multiple samples pooled together)
  • isolate RNA using Qiagen RNeasy Plant Mini Kit
  • elute RNA into the final volume of 50 uL


<DNase I treatment> TURBO DNA-free™ Kit (Ambion AM1907)

  • To 50 uL RNA solution
  • Add 5 uL of 10xbuffer
  • Add 1 uL of Dnase I enzyme sol.
  • Mix gently
  • Incubate at 37C for 20 min
  • Add 5 uL of inactivation buffer
  • Incubate at RT for 2 min
  • Mix it every minute
  • Spin down for 2 min at 13,000rpm
  • Remove 40 uL supernatant (avoid bottom pellet).


<RNA concentration determination>

  • Take 3 uL and mix with 297 uL of ddH2O (100 times dilution)
  • Freeze the rest ------------------------------------------------------------------à store at –80C
  • Measure A260nm, 280nm, 230nm
  • Calculate RNA conc.(ug/uL) = A260 x 40 x 100 (dilution factor) / 1000
  • A260/A280 > 1.8
  • A260/A230 > 1.8


<cDNA synthesis>

  • High Capacity cDNA RT Kit (Applied Biosystem, 4368813) [0.5ug RNA/50uL Rx]
    • 10.5 uL Nuclease Free water
    • 5 uL 10 x RT buffer
    • 2 uL 25 x dNTPs
    • 5 uL 10 x random primer
    • 2.5 uL RT enzyme
    • +
    • 25 uL of RNA solution (=0.5 ug) (make 2 ug/100uL RNA solution)
    • 50 uL total volume
    • “RT-FLO” method
    • 25 C 10 min
    • 37 C 2 hr
    • 4 C forever


<Primer efficiency test>

  • To check if efficiency of primers used are 100 ± 10 % (especially for Ct experiment)
  • Dilute cDNA 5 folds for 5 times. (highest cDNA is typically 10 fold diluted)
  • Run qPCR using each primer set
  • If the slop is close to – 0.33, efficiency of primers are 100 %
    • 5 uL Fast SYBR Green mix
    • 1.5 uL F primer (2 uM) = 300 nM final conc.
    • 1.5 uL R primer (2 uM) = 300 nM final conc.
    • 2 uL Template diluted cDNAs
    • 10 uL total


<Internal std primer test (for Ct experiment)>

  • To check if Ct value for internal primer (e.g. ACTIN) is within ± 1 cycle among different cDNA samples.
    • 5 uL Fast SYBR Green mix
    • 1.5 uL F primer (2 uM) = 300 nM final conc.
    • 1.5 uL R primer (2 uM) = 300 nM final conc.
    • 2 uL Template 1/50 diluted cDNA (400 pg RNA derived)
    • 10 uL total


<qPCR set up>

  • Open “StepOne Plus” à “advance set up”


<Data analysis>

  • Set baseline
  • Set threshold for each target gene
  • Check melting curve
  • Export data to Excel file
  • Copy slides to paint and save
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