The technique of small interfering RNA (siRNA) is revolutionizing scientific research by enabling researchers to knock-down a gene message and subsequent protein levels. Using siRNA, researchers can conduct cellular-based assays in the absence or presence of a targeted gene’s protein. Such experiments are invaluable to molecular discovery and have begun to replace gene knock-out, chemical inhibition of protein activity and other traditional methodologies for gene characterization. As a result, many companies have come out with kits to simplify the processes of siRNA as an everyday tool. Ambion is one such company, introducing not only pre-designed siRNAs, control siRNAs and siRNA transfection reagents, but also siRNA labeling kits.
The Silencer siRNA Fluorescein (FAM) Labeling Kit from Ambion is a great complement to siRNA procedures because it allows you to label up to 65 ug of siRNA without affecting its biological function. The labeled siRNA can then be used to visually determine transfection efficiency, siRNA subcellular localization and stability. I have used this kit extensively and have obtained efficient detection of transfected siRNA, as well as, easy determination of cell stability in target lines. As a result, I have saved considerable time avoiding Northern Blot analysis of knock-down efficiency to optimize transfection or determine siRNA stability. Instead, I have used these lengthy procedures for follow-up confirmation of my results alone.
This kit is fairly straight-forward and easy to use. However, scaling up the reaction volume to label a large quantity of siRNA can lead to inefficient labeling. To avoid this problem, I use smaller reaction volumes (50 ul total volume of assembled labeling mix or 5 ug of 21 mer duplex siRNA) for the labeling procedure. After performing the labeling reaction, ethanol precipitation and re-suspension, I pool the aliquots before determining labeled RNA concentrations using Ambion’s online concentration calculator. Performing the reaction in this manner has given me a high yield of labeled product with consistent results for several different siRNAs.
One downfall of this kit is that you must be very careful to perform all of the reaction steps in the dark to ensure a good yield. This can become quite time consuming when you must label a large quantity of siRNA, since the labeling must be done in smaller aliquots. The smaller aliquots must be covered well with foil, kept sterile and incubated with fairly precise timing. This kit is a worthwhile investment because it allows you to visualize siRNA transfection efficiency, localization and stability without lengthy procedures like Northern Blotting, thus, it ultimately saves time and reagents. Therefore, despite the intricacies of the kit procedures, Ambion’s Silencer siRNA Labeling Kit is a good purchase for both beginners and those who are more advanced in working with siRNA technology.
Amy Bellmeyer, M.S.
Research Technician
Northwestern University
Division of Pulmonary and Critical Care Medicine