X-tremeGENE™ HP DNA Transfection Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Do not aliquot X-tremeGENE HP DNA Transfection Reagent; store in the original glass vials.

Upstream tips
Do not aliquot X-tremeGENE HP DNA Transfection Reagent; store in the original glass vials.

Publication protocol

Cell Transfection and Dual-luciferase Reporter Assay
Cells were seeded in 24-well tissue culture plates (CELLTREAT Scientific Products, LLC, Shirley, MA, USA) at a density of 150,000 cells/mL (48 hour assays) or 100,000 cells/mL (96 hour assays) in a volume of 500 µL/well. Twenty-four hours after seeding, test plasmids (constructs or empty vector) were transfected into cells using 1∶1 ratio of X-tremeGENE HP DNA transfection reagent (µL) (Roche USA, Indianapolis, IN, USA) to test plasmids (µg). Co-transfection with miRNAs (Vana® miRNA mimic, Invitrogen Corporation, Carlsbad, CA, USA) was performed using 5∶2∶1 ratio of X-tremeGENE siRNA transfection reagent (µL) (Roche USA, Indianapolis, IN, USA), to test plasmids (µg) and miRNA (µg). GIBCO Opti-MEM ® 1 media (Invitrogen Corporation, Carlsbad, CA, USA) was used as serum-free diluent. As a control for transfection, a renilla luciferase plasmid, (pRL-CMV, Promega Corporation, Madison, WI, USA) was co-transfected (0.2 pg/µL) with each construct or empty vector. An empty pGL3 vector and a random sequence miRNA molecule (Vana™ miRNA mimic, Negative Control #1, Invitrogen Corporation, Carlsbad, CA, USA) that has been validated to produce no identifiable effects on known miRNA function (control miR) were used as control of luciferase expression. Cells were maintained for another 48 hours or 96 hours (undifferentiated and differentiated cells) before harvesting and assaying for luciferase activity. The dual-luciferase reporter assay system (Promega Corporation, Madison, WI, USA) was used to assess gene expression, per manufacturer’s instructions. Two different maxi-preps were tested on at least two different days for each construct as well as for the “empty” pGL3 (no insert).

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for X-tremeGENE™ HP DNA Transfection Reagent below.

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