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AlanFung:LabNotes/Reprogramming/2010-11-10
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==one step qRT-PCR== *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 {| {{table}} | ||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 {| {{table}} | ||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 {| {{table}} | ||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 βone step qrt_pcr" *50C 3min -> hold -> 95C 5min -> hold (95C 15sec -> 60C 30sec) x 40 cycle -> 40C 1min [[File:SPEN_RTPCR_Trim.jpg|500px]] *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
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