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
- 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