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X tremegene sirna transfection kit

Manufactured by Roche
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The X-tremeGENE siRNA Transfection Kit is a laboratory tool used for the delivery of small interfering RNA (siRNA) into cells. It facilitates the introduction of siRNA into the target cells, enabling the study of gene function through RNA interference (RNAi) technology.

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Lab products found in correlation

Synthetic pre mir206, by Thermo Fisher Scientific (2 mentions) Nerve growth factor (ngf), by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Npc1 sirna, by Qiagen (1 mentions) Glass bottom microwell dishes, by MatTek (1 mentions) Horse serum, by Merck Group (1 mentions) Pc12 cells, by American Type Culture Collection (1 mentions) Rpmi 1640 medium, by Merck Group (1 mentions) Eclipse te2000 s, by Nikon (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

X-tremeGENE siRNA X-tremeGENE Kit X-tremeGENE SiRNAs

6 protocols using x tremegene sirna transfection kit

1

Transfection of C2C12 Cells with let-7e-5p

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C2C12 cells (a mouse myoblast cell line; KAC Co. Ltd., Kyoto, Japan), were plated in 24-well plates and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (day 2). The medium was changed every other day. When the cells reached 80% confluence, they were differentiated in DMEM supplemented with 2% horse serum (differentiation medium) (day 0). At 24 h after the medium change, the cells were transfected with 30 nM let-7e-5p mimic/inhibitor or a scrambled sequence (mirVana ®), which were purchased from Thermo Fisher Scientific, using X-treme Gene siRNA transfection kit (Roche, Mannheim, Germany) according to the manufacturer’s recommendations (day 1). At 96 h (day 5) post-transfection, the myotube cells were evaluated through various experiments.
Okamura T., Okada H., Hashimoto Y., Majima S., Senmaru T., Nakanishi N., Asano M., Yamazaki M., Hamaguchi M, & Fukui M. (2021). Let-7e-5p Regulates IGF2BP2, and Induces Muscle Atrophy. Frontiers in Endocrinology, 12, 791363.
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2

Luciferase Assay for miR-206 Target Validation

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Details of these experiments are given in the Supplementary material. In short, a 52 base pair putative miR-206 target sequence from the 3′ UTR region of LXR, or a scrambled negative control was cloned using PremiR-Reporter constructs and synthetic Pre-miR206 from Ambion and the expression plasmid for miR206 from SBI (System Biosciences, PA, USA).
For the luciferase assay, 100 ng/ml of the PremiR-Reporter construct was co-transfected with 25 ng/ml β-Gal control plasmid and also with the 100 nM synthetic precursor-miR206 strand or the 1 μg/ml of miR-206 expression vector into COS-7 cells using X-tremeGENE siRNA Transfection Kit (Roche). After 36 h, cells were lysed and 50 μl of the lysate was used for β-Gal assay and 10 μl (2×) was used for luciferase assay carried out using the manufacturer’s protocol.
Vinod M., Chennamsetty I., Colin S., Belloy L., De Paoli F., Schaider H., Graier W.F., Frank S., Kratky D., Staels B., Chinetti-Gbaguidi G, & Kostner G.M. (2014). miR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages. Biochimica et Biophysica Acta, 1841(6), 827-835.
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3

Validation of miR-206 Regulation of LXR

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Details of these experiments are given in the Supplementary material. In short, a 52 base pair putative miR-206 target sequence from the 3′UTR region of LXR, or a scrambled negative control was cloned using PremiR-Reporter constructs and synthetic Pre-miR206 from Ambion and the expression plasmid for miR206 from SBI (System Biosciences, PA, USA).
For the luciferase assay, 100 ng/ml of the PremiR-Reporter construct was co-transfected with 25 ng/ml β-Gal control plasmid and also with the 100 nM synthetic precursor-miR206 strand or the 1 μg/ml of miR-206 expression vector into COS-7 cells using X-tremeGENE siRNA Transfection Kit (Roche). After 36 h, cells were lysed and 50 μl of the lysate was used for β-Gal assay and 10 μl (2 ×) was used for luciferase assay carried out using the manufacturer's protocol.
Vinod M., Chennamsetty I., Colin S., Belloy L., De Paoli F., Schaider H., Graier W.F., Frank S., Kratky D., Staels B., Chinetti-Gbaguidi G, & Kostner G.M. (2014). miR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages. Biochimica et Biophysica Acta, 1841(6), 827-835.
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4

siRNA Knockdown of Tead1 in VSMC

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We performed siRNA transfection using X-tremeGENE siRNA transfection kit (Roche; Basel, BS, Switzerland) according to the manufacturer’s protocol. Briefly, VSMC was transfected with target-specific Tead1 siRNA (Tead1i; GenePharma, Shanghai, China) and incubated with 10 μL X-tremeGENE siRNA transfection reagent. Scramble siRNA was used as a nonspecific control. After 8 hours of transfection, VSMC was refreshed with the complete medium. The sequence of Tead1i was shown as following: Tead1i (5’-GGCAGATAAGCCGATTGACAACG-3’).
Sun X., Li S., Gan X., Chen K., Yang D, & Yang Y. (2020). NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells. Aging (Albany NY), 12(10), 9726-9744.
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5

Dissecting Neuronal Differentiation and Npc1 Knockdown in PC-12 Cells

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PC-12 cells (CRL1721) were purchased from ATCC and cultured at 37 °C in a humidified atmosphere containing 5% CO2 in RPMI-1640 medium supplemented with 10% horse serum (Sigma, St. Louis, MI) and 5% fetal bovine serum (Invitrogen, Carlsbad, CA). Cells were seeded in Poly-L-lysine (P1524, Sigma) coating glass bottom microwell dishes (MatTek Corporation, Ashland, MA) for two days before differentiation with nerve growth factor (300 ng/ml, Sigma) for 10 days. Npc1 siRNA (Strauss et al., 2010 (link)) (100 pmol, Qiagen, Valencia, CA) and scrambled control siRNA were transfected together with a GFP plasmid (2 μg) immediately before differentiation using the X-tremeGENE-siRNA transfection kit according to the manufacturer instructions (Roche). MMP-12 inhibitor (MMP408, 500 nM, Abcam) (Li et al., 2009a (link); Li et al., 2009b (link)) was added 4 h after transfection. Nine days after transfection, cells were fixed with 2% PFA for 15 min; cells were then examined and imaged using a fluorescence microscope (Nikon Eclipse TE2000-S) and a DS camera from Nikon. Neuritic length was measured as previously described using NIH ImageJ software (Das et al., 2004 (link)). Data were expressed as means of four sets of individual experiments; for each experiment at least 25–26 cells per group were analyzed and averaged values were used to calculate group means.
Liao G., Wang Z., Lee E., Moreno S., Abuelnasr O., Baudry M, & Bi X. (2015). Enhanced expression of matrix metalloproteinase-12 contributes to Npc1 deficiency-induced axonal degeneration. Experimental neurology, 269, 67-74.
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6

Knockdown of ENSMUST00000117266 in Neonatal Mouse Cardiomyocytes

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We performed siRNA transfection using X-tremeGENE siRNA transfection kit according to the manufacturer’s protocol (Roche). Briefly, neonatal mouse cardiomyoyctes were transfected with the mixture of three different target-specific ENSMUST00000117266 siRNAs (GenePharma) and 10 μl X-tremeGENE siRNA transfection reagent. For ASO transfection, neonatal mouse cardiomyoyctes were transfected with the mixture of ENSMUST00000117266 ASOs (Gene Pharma) modified with phosphorothioates and locked nucleic acid using lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen). Scramble siRNA or ASO was used as a nonspecific control. After 8 hours of transfection, cells were refreshed with the complete medium and cultured for another 48 hours before harvesting for RNA extraction. The sequences of siRNAs and ASOs were shown in Table 2.
Sun X., Han Q., Luo H., Pan X., Ji Y., Yang Y., Chen H., Wang F., Lai W., Guan X., Zhang Q., Tang Y., Chu J., Yu J., Shou W., Deng Y, & Li X. (2017). Profiling analysis of long non-coding RNAs in early postnatal mouse hearts. Scientific Reports, 7, 43485.
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