HiroshiMaeda:qPCR

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

HiroshiMaeda Protocol: 2011.2.20 last updated


 * 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.


 * 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

 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).


 * 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


 * 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
 * 37 C	2 hr
 * 4 C	forever


 * 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


 * 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


 * 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