The plasmid pCMV-MSI2 (pMSI2) was generated by GeneCopoeia, and Flag-tagged KLF4 (Flag-KLF4) was described previously (22 (link)). Small interfering RNAs (siRNAs) for MSI2 (siMSI2#1, 5'-gcgaacacaguaguggaagauguaa-3'; siMSI2#2, 5'-uuacaucuuccacuacuguguucgc-3') were synthesized by Life Technologies (2 (link), 4 (link)), and KLF4 siRNAs consisted of pools of three to five target-specific 19- to 25-nt siRNAs designed to knock down KLF4 expression (Santa Cruz Biotechnology) as demonstrated in our previous studies (8 (link)). A negative control siRNA (Invitrogen) and control pCMV vector were used. Transfection of plasmids and siRNAs into PDAC cells was performed using Lipofectamine 2000 CD transfection reagent (Invitrogen). For transient transfection, cells were transfected with plasmids or siRNA at different doses as indicated for 48 hours before performance of functional assays. PDAC cells treated with transfection reagent alone were included as mock controls.
Protocol detail
Plasmid Transfection and siRNA Knockdown in PDAC Cells
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Assays
Cells
Functional performance
Klf4
Lipofectamine 2000
Pdac
Plasmids
Sirna
Transfection
Transient
Corresponding Organization : Shanghai East Hospital
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Variable analysis
independent variables
- PCMV-MSI2 (pMSI2) plasmid
- Flag-tagged KLF4 (Flag-KLF4)
- Small interfering RNAs (siRNAs) for MSI2 (siMSI2#1, siMSI2#2)
- KLF4 siRNAs
- Transfection of plasmids and siRNAs into PDAC cells using Lipofectamine 2000 CD transfection reagent
- Functional assays performed 48 hours after transfection
- Negative control siRNA (Invitrogen)
- Control pCMV vector
- PDAC cells treated with transfection reagent alone (mock controls)
- Negative control siRNA (Invitrogen)
- Control pCMV vector
- PDAC cells treated with transfection reagent alone (mock controls)
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