SYBR vs' Taqman

1
SYBR vs. Taqman

• Taqman
• Dual labelled fluorescent oligo probe, uses FRET
• 300/probe (Operon)
• Highly specific for target
• Less optimization
• few targets many assays

• SYBR green
• Fluorescent dye binds to all dsDNA
• Less expensive
• Non-specific
• More optimization
• Many targets few assays

Samples analysed independently by both methods
gave similar results
2
Materials needed

• cDNA RNA must be of high quality and free of DNA
  contamination, reversed transcribe as usual with
  random hexamer primers.
• Universal master mix purchased from ABI,
  specific for SYBR or Taq man. (400/400rxn)
• Primers and Taq man probe designed using primer
  express software, span an intron if possible.
• Housekeeping gene primers/probe ex. GAPDH,
  18srRNA (best). Can be purchased from ABI, or
  user designed
• Plastic ware ABI, Optically clear 96 well
  plates and adhesive covers. Lots of pipette
  tips!

3
Preliminary experiments

• Determine optimal primer concentration
• NEED to do this for SYBR green assays
• Assay positive sample with varying primer
  concentrations (50, 150, 300, 900nM ea.)
• Optimal concentration gives lowest Ct value, no
  primer dimers or non-specific amplification.
  (melt curve analysis)

4
Preliminary experiments

• Standard curve validation
• To determine if PCR reaction kinetics for the
  target are comparable to that of endogenous
  control.
• Run ten fold dilutions of known positive sample
  with new target as well as endogenous ctrl
  primers (5 dilutions, each point in
  triplicate)
• Plot dCt vs log conc. slope lt0.1validated
• If method validates, samples can be compared
  directly to endogenous control without a standard
  curve, using Ct values.
• Otherwise, you have to set up a standard curve
  each time, and compare quantities of cDNA.

5
Finally, the experiment!

• With or without standard curve
• Each sample paired to endogenous ctrl
• Each sample in triplicate
• (this does not mean n3!)
• No template control
• checks for contamination