HiroshiMaeda:qPCR

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”

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