DLD-1 and HCT116wt cells were seeded in 6-well plates (6×105 cells with 2 ml medium per well) and cultured at 37°C for 24 h. A mixture containing 110 pmol Noxa siRNA (cat. no. sc-37305; Santa Cruz Biotechnology, Inc.) or control siRNA (cat. no. sc-36869; Santa Cruz Biotechnology, Inc.), 200 µl jetPRIME® buffer (Polyplus-transfection SA) and 4 µl jetPRIME® siRNA transfection reagent (Polyplus-transfection SA) was vortexed for 10 sec, spun down and incubated for 10 min at room temperature. The mixture was subsequently added to each well of the 6-well plate and incubated at 37°C for 24 h. Cells were then harvested for further experiments.
Jetprime sirna transfection reagent
Manufactured by Polyplus Transfection
Sourced in United States
JetPRIME siRNA transfection reagent is a cationic polymer-based transfection reagent designed for efficient and consistent delivery of small interfering RNA (siRNA) into a variety of cell types. It facilitates the transfer of siRNA molecules into the target cells, enabling gene silencing studies.
Automatically generated - may contain errors
9 protocols using jetprime sirna transfection reagent
1
Noxa siRNA Transfection in DLD-1 and HCT116 Cells
2
Transfection and miR-200c Modulation
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4T1 cells were transfected with 50 µM Mircury scramble or miR-200c LNA-oligonucleotides (Exiqon) using jetPRIME-siRNA Transfection reagent (Polyplus) according to manufacturer’s instructions. After 16 h, cells were incubated with fresh medium for 32 h and then treated with AA6 or water, as control, for additional 24 h.
3
Insulin Signaling Pathway Regulation
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HEK-293, MEFs and MCF7 cells were grown in DMEM (1:1) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL Penn-strep. For the starvation and insulin stimulation experiments, cells were grown in serum-starved DMEM media for overnight (16 h) prior to stimulation with 1 μM insulin for 15 min. For the pharmacological inhibitions, 16 h serum-starved cells were treated 2 h with DMSO (as a vehicle) or ERK inhibitor U0126 (10 μM), or Akt inhibitor MK2206 (10 μM) or PI3K inhibitor LY294002 (50 μM) or mTORC1 inhibitor rapamycin (300 nM) prior to stimulation with 1 μM insulin for 15 min. For knockdown of ERK1/2, cells were transfected with siRNA of ERK1/2 for 56 h prior to 16 h serum starvation, followed by stimulation with 1 μM insulin for 15 min. Cells were transfected with siRNA of ERK1/2 using the jetPRIME™ siRNA Transfection Reagent (Polyplus transfection).
4
Investigating Cell Migration and Adhesion via α2,6-Sialylation
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To investigate the effect of α2,6‐sialylation on cell migration and cell‐cell adhesion, two approaches were used: (1) treatment of cells with SNA (1:100, L‐1300; Vector Labs), a lectin that can specifically bind to α2,6‐linked sialic acid residues, to block the role of α2,6‐linked sialic acids. (2) siRNA‐mediated knockdown of ST6GAL1 to decrease the levels of α2,6‐linked sialic acids. ST6GAL1 siRNA (si‐ST6GAL1) oligonucleotides (sense: 5′‐CAGCCAACUUCCAACAAGAdTdT‐3′/antisense: 5′‐dTdTGUCGGUUGAAGGUUGUUCU‐3′) and non‐targeting control siRNA (si‐NC) oligonucleotides (sense: 5′‐UUCUCCGAACGUGUCACGUTT‐3′/ antisense: 5′‐ACGUGACACGUUCGGAGAATT‐3′) were synthesized by GenePharma. Briefly, Ishikawa cells were transfected with a final concentration of 110 pmol (for a 6‐well plate) or 55 pmol (for a 12‐well plate) of si‐ST6GAL1 and si‐NC oligonucleotides using jetPRIME siRNA transfection reagent (114–15, Polyplus‐transfection® SA) according to the manufacturer's instructions. Quantitative RT‐PCR (qRT‐PCR) and Western blot were performed after cells were transfected with si‐ST6GAL1 or si‐NC for 72 h.
5
siRNA Knockdown of PRMT1 and PRMT5
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The siRNAs were synthesized by Boshang. The small interfering RNA (siRNA) duplexes used for the controls have been previously described [34 (link)]. The PRMT5 siRNA duplexes target the sequences 5’-GCCCAGUUUGAGAUGCCUU-3′ (#1) and 5’-CCGCUAUUGCACCUUGGAA-3′ (#2). The PRMT1 siRNA duplexes target the sequences 5’-CCACCAGCCCCGAGUCCCC-3′ (#1) and 5’-ACCGCAACUCCAUGUUUCA-3′ (#2). The negative control was 5’-UUCUCCGAACGUGUCACGU-3′. The siRNA transfections were carried out with the jet PRIME® siRNA Transfection Reagent (Polyplus) following the manufacturer’s instructions.
6
Silencing MT1 and MT2 in BMMs
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The pre-designed siRNA duplexes for MT1, MT2, and the universal negative control were purchased from GenePharma (Shanghai, China). Oligonucleotide siRNA duplexes were transfected into BMMs with jetPRIME siRNA transfection reagent (Polyplus, New York, NY, USA) according to the manufacturer’s instructions. The efficiency of transfection was >80% when measured by FAM-labeled siRNA control transfection.
7
CYTSA Depletion in Colorectal Cancer Cells
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One or two different siRNAs (5′-GAACUGAACAGCCAGGAUA [dT][dT]-3′ and 5′-ACAGAAGGCTATCAGAATA[dT][dT]-3′) that targeted human CYTSA were used to deplete CYTSA in CRC cells. A validated non-targeting siRNA (Sigma-Aldrich, St. Louis, MO, USA) was used as a control [15 (link)]. The jetPRIME siRNA transfection reagent (Polyplus, New York, NY, USA) was used according to the manufacturer’s instructions. In brief, the siRNA and jetPRIME reagent were mixed together, incubated for 15 min, and added to CRC cells grown in cell culture medium without antibiotics in six-well plates. After 24 h, cells were washed with phosphate-buffered saline (PBS) and cultured with regular cell culture media with antibiotics. Forty-eight hours from the start of transfection, cells were lysed with either (1) TRIzol® to isolate RNA or (2) radioimmunoprecipitation assay (RIPA) buffer to extract proteins for further analyses.
8
Cellular Fixation and siRNA Transfection
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Water was purified with a Millipore Q-POD apparatus. The paraformaldehyde (PFA 16% solution) and the glutaraldehyde (25% solution) solutions were of Electron Microscopy quality grade and purchased from Electron Microscopy Sciences. The jet PRIME siRNA transfection reagent was from PolyPlustransfection. Other chemical reagents and solvents were obtained from commercial sources (Sigma Aldrich, Carl Roth,
9
Silencing TGM2 Gene Expression
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The siRNA duplexes oligonucleotides of TGM2 and the letivirus against Tgm2 were purchased from Gene Pharma (Shanghai, China). The siRNA duplexes oligonucleotides were: si-TGM2-1 5'-GCCUGAUCCUUCUAGAUGUTT-3', si-TGM2-2, 5'-GUCGUGACCAACUACAACUTT-3', si-TGM2-3, 5'-GGCUGAAGAUCAGCACUAATT-3' . The siNC does not have any target sites in human genome. JetPRIME siRNA transfection reagent (Polyplus transfection) was used to transfect siRNA according to the manufacturer's instructions. The lentivirus particles were transfected into cells in the presence of lipofectamine 2000 (Invitrogen, Carlbad, CA, USA) according to the manufacturer's instructures, when cells confluence grew up to 50%. Then the virally infected cells were treated with 2 μg/ml puromycin (Gibco, A1113802, USA) for 7 days to select stable knockdown cells. The sequence targeting Tgm2 were: sh-Tgm2-1, 5'-CGGCTGTCAGCAAGTGAAGTA-3', sh-Tgm2-2, 5'-GAGTGGTGACCAACTACAACT-3'.
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