siRNA / miRNA gene silencing Rat - MTLn3 β1 integrin/Itgb1

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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ON-TARGETplus Rat Itgb1 (24511) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	For live-cell imaging, 105 MDA-MB-231 cells were plated on a six-well plate and then transfected with 0.5 μg of TagRFP-cortactin (Oser et al., 2009) and GFP-Tks5 (kindly provided by Sara Courtneidge, Burnham Institute for Medical Research, La Jolla, CA; Stylli et al., 2009) using Lipofectamine LTX and PLUS reagent 24 h before imaging per manufacturer's instructions. MT1-MMP-GFP was described previously (Galvez et al., 2002). The control empty vector and stable β1 integrin shRNA cell lines were generated using pGIPZ shRNA lentiviral vectors (Open Biosystems, Huntsville, AL). ON-TARGETplus SMARTpool β1 integrin (human, L-004506-00–MDA-MB-231 cells; rat, L-089600-01–MTLn3 cells), β3 integrin (L-004124-00-0010), Arg (L-003101-00), and cofilin (L-012707-00) siRNAs were obtained from Dharmacon (Lafayette, CO). The siGENOME β1 integrin siRNA (D-004506-03) was also from Dharmacon. Nonsilencing control siRNA was obtained from Qiagen (Valencia, CA). For siRNA transfection, 106 MDA MB-231 cells were transfected with 2 μM siRNA using the Amaxa V nucleofection system (Lonza, Basel, Switzerland) 48–96 h before each experiment. MTLn3 cells were transfected with 100 nM β1 integrin siRNA in Oligofectamine for 48 h as described previously (Kempiak et al., 2005).

Protocol tips
For live-cell imaging, 105 MDA-MB-231 cells were plated on a six-well plate and then transfected with 0.5 μg of TagRFP-cortactin (Oser et al., 2009) and GFP-Tks5 (kindly provided by Sara Courtneidge, Burnham Institute for Medical Research, La Jolla, CA; Stylli et al., 2009) using Lipofectamine LTX and PLUS reagent 24 h before imaging per manufacturer's instructions. MT1-MMP-GFP was described previously (Galvez et al., 2002). The control empty vector and stable β1 integrin shRNA cell lines were generated using pGIPZ shRNA lentiviral vectors (Open Biosystems, Huntsville, AL). ON-TARGETplus SMARTpool β1 integrin (human, L-004506-00–MDA-MB-231 cells; rat, L-089600-01–MTLn3 cells), β3 integrin (L-004124-00-0010), Arg (L-003101-00), and cofilin (L-012707-00) siRNAs were obtained from Dharmacon (Lafayette, CO). The siGENOME β1 integrin siRNA (D-004506-03) was also from Dharmacon. Nonsilencing control siRNA was obtained from Qiagen (Valencia, CA). For siRNA transfection, 106 MDA MB-231 cells were transfected with 2 μM siRNA using the Amaxa V nucleofection system (Lonza, Basel, Switzerland) 48–96 h before each experiment. MTLn3 cells were transfected with 100 nM β1 integrin siRNA in Oligofectamine for 48 h as described previously (Kempiak et al., 2005).

Protocol tips

For live-cell imaging, 105 MDA-MB-231 cells were plated on a six-well plate and then transfected with 0.5 μg of TagRFP-cortactin (Oser et al., 2009) and GFP-Tks5 (kindly provided by Sara Courtneidge, Burnham Institute for Medical Research, La Jolla, CA; Stylli et al., 2009) using Lipofectamine LTX and PLUS reagent 24 h before imaging per manufacturer's instructions. MT1-MMP-GFP was described previously (Galvez et al., 2002). The control empty vector and stable β1 integrin shRNA cell lines were generated using pGIPZ shRNA lentiviral vectors (Open Biosystems, Huntsville, AL). ON-TARGETplus SMARTpool β1 integrin (human, L-004506-00–MDA-MB-231 cells; rat, L-089600-01–MTLn3 cells), β3 integrin (L-004124-00-0010), Arg (L-003101-00), and cofilin (L-012707-00) siRNAs were obtained from Dharmacon (Lafayette, CO). The siGENOME β1 integrin siRNA (D-004506-03) was also from Dharmacon. Nonsilencing control siRNA was obtained from Qiagen (Valencia, CA). For siRNA transfection, 106 MDA MB-231 cells were transfected with 2 μM siRNA using the Amaxa V nucleofection system (Lonza, Basel, Switzerland) 48–96 h before each experiment. MTLn3 cells were transfected with 100 nM β1 integrin siRNA in Oligofectamine for 48 h as described previously (Kempiak et al., 2005).

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