AlanFung:LabNotes/Reprogramming/2010-11-10

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one step qRT-PCR[edit]

  • The purpose of this experiment is to perform quantitative RT-PCR for the detection of the efficiency of SPEN gene knockdown.


  • Superscript III RT/Platinum Taq Mix 1ul
  • 2X SYBR Green Reaction Mix 25ul
  • Forward Primer, 10uM 1ul
  • Reverse Primer, 10uM 1ul
  • Template (1pg to 1ug total RNA) ul
  • DEPC-treated water to 50ul


  • Template volume to get 1ug of RNA
24 48
14 1.44 1.21
15 1.32 1.43
93 1.33 1.73
94 1.02 1.76
95 1.43 1.95
96 1.13 2.43
  • H2O volume
24 48
14 20.56 20.79
15 20.68 20.57
93 20.67 20.27
94 20.98 20.24
95 20.57 20.05
96 20.87 19.57
  • Prepare Mastermix
13 13
Superscript III RT/ Platinum Taq Mix 1 13
2X SYBR Green Reaction Mix 25 325
Forward Primer (10uM) 1 13
Reverse Primer (10uM) 1 13


  • Running at Program “one step qrt_pcr"
  • 50C 3min -> hold -> 95C 5min -> hold (95C 15sec -> 60C 30sec) x 40 cycle -> 40C 1min

File:SPEN RTPCR Trim.jpg

  • Ct value ranges from ~16-17, all samples including the negative controls shows the amplification
  • I was not expecting an amplification on the samples that were knocked down by the 4 constructs, since the transient knockdown by shRNA should alter the gene expression resulting in a decrease in expression and here I am not seeing a significant difference between the negative control, the 24hr knock down and the 48hr knock down.
  • Origene tech support suggests running western blot assay, as it is a more direct way to detect the knockdown, yet it will be difficult to do so, for the protein size is very large and will be hard to blot.
  • Emailed Jessica for advice