On targetplus sicontrol non targeting sirna

Specific silencing of VRK1 was performed using siVRK1-02 (siV1-02), designed with the SMARTselection algorithm (Dharmacon, Lafayette, CO) and obtained from Dharmacon (DHARMACON RNA Technologies). The sequence target of siVRK1-02 is 5'-CAAGGAACCTGGTGTTGAA-3'. As negative control, the “ON-TARGETplus siCONTROL Non-targeting siRNA” from DHARMACON was used. The efficiency of RNAi transfection was determined with “siGLO RISC-free siRNA” (DHARMACON) labeled with a red fluorochrome [21 (link)].
Cells were transfected with the indicated siRNA at a concentration of 20 nM using Lipofectamine 2000 Reagent” (Invitrogen, Carlsbad, CA) according to manufactures instructions. After transfection, cells were processed for specific experiments as previously reported [14 (link), 17 (link), 24 (link)].

Specific silencing of VRK1 was performed using two different siRNA from Dharmacon (DHARMACON RNA Technologies). The VRK1 sequences targeted by these siRNA oligonucleotides were siVRK1-02 (siV1-02), CAAGGAACCTGGTGTTGAA; and siVRK1-03 (siV1-03): That GGAAUGGAAAGUAGGAUUA. As negative control, the “ON-TARGETplus siCONTROL Non-targeting siRNA” from DHARMACON was used and is indicated as siCt in experiments. The efficiency of RNAi transfection was determined with “siGLO RISC-free siRNA” (DHARMACON). Briefly, cells were transfected with the indicated siRNA at a concentration of 20 nM using Lipofectamine 2000 Reagent (Invitrogen) or Lipotransfectin (Nivorlab) [18 (link),34 (link),74 (link)]. After transfection, cells were processed at the times indicated in specific experiments that were performed as previously reported [19 (link),77 (link)]. The depletion of VRK1 by siRNA has been previously reported for A549 [18 (link),21 (link),36 (link)] and HT144 [20 (link),21 (link)] cell lines.

VRK1 knockdown was performed using three different siRNA specific for VRK1 (Accession number NM_003384): siVRK1-01 (siV-01), siVRK1-02 (siV-02), siVRK1-03 (siV-03) and siVRK1-09 (siV-09) (all from Dharmacon RNA Technologies- GE Healthcare). The sequences targeted by these VRK1 siRNA oligonucleotides were siVRK1-01: GAAAGAGAGTCCAGAAGTA; siVRK1-02: CAAGGAACCTGGTGTTGAA; si-VRK1-03: GGAAUGGAAAGUAGGAUUA; and siVRK1-09: AGGUGUACUUGGUAGAUUA. As negative control the “ON-TARGETplus siCONTROL Non-targeting siRNA” (siCt) (Dharmacon) was used. The efficiency of RNAi transfection was determined with “siGLO RISC-free siRNA” (DHARMACON) labelled with a red fluorochrome. All of them have been previously used34 (link)48 (link)51 (link).
Briefly, cells were transfected with the indicated siRNA at a concentration of 20 nM using Lipofectamine 2000 Reagent” (Invitrogen) according to manufacturer instructions. After transfection cells were processed for specific experiments at the times indicated in them. For rescue experiments, cells were transfected with the indicated siRNA using Lipofectamine 2000 Reagent, and 36 hours later, cells were retransfected with plasmids using JetPI reagent (Poly Plus, Ilkirch, France) according to manufacturer instructions. Targeted protein and plasmid expression were analysed thirty-six hours after the second transfection51 (link).