X-tremeGENE™ siRNA Transfection Reagent Protocol & Troubleshooting

Product No. SITRAN-RO

Protocol

siRNA in Primary Lung Fibroblasts Transfection

Roche has no data for this application, but there is a report from a customer, briefly described below.

Wild rat lung fibroblasts were successfully cotransfected with a plasmid containing the fibroblast growth factor 2 (FGF2) and either a non-specific siRNA, against luciferase GL2, or a specific siRNA against FGF2. The ratio used was 9 µL of X-tremeGENE™ siRNA Transfection Reagent to 1 µg of nucleic acid.
Cells were harvested 66 hours after transfection and analyzed by western blotting. The level of expression of ß-actin was used as the loading control.
Samples transfected with siRNA GL2 did not decrease the level of expression of FGF2 and ß-actin. FGF2-specific siRNA induced a 45% decrease of the level of expression of FGF2.
These results show that X-tremeGENE™ siRNA Transfection Reagents can be used to efficiently transfect (and cotransfect) plasmids and siRNAs into primary cells.

siRNA, Reverse TR and Expression Plasmids on Transfected Cell Microarrays

It is possible to transfect siRNA and expression plasmids on transfected cell microarrays using the X-tremeGENE™ siRNA Transfection Reagent.

The details of the experimental protocol are provided in this Biochemica article: Efficient Reverse Transfection of both siRNA and Expression Plasmids on Transfected Cell Microarrays Using X-tremeGENE™ siRNA Transfection Reagent.

Troubleshooting

Precipitates

When adding DNA, there may be precipitates which are not due to X-tremeGENE™ siRNA Transfection Reagent. The formation of precipitates may be due to a high concentration of salt in the DNA sample. Roche recommends purifying DNA using Genopure Plasmid Kits.