ON-TARGETPlus™ siRNA reagents From Dharmacon, Inc.

While siRNA technology has been around since the start of the decade, two key factors have remained constant in determining its usefulness in laboratory and clinical settings; namely, sequence design and product delivery. Dharmacon has emerged (alongside its competitors, Ambion and Invitrogen) as a prominent resource in RNA-related research, supplying a variety of experimentally-viable RNA products ranging from mRNA to one of its newest and most demanded products: ON-TARGETPlus™ siRNA. Compared to the more basic siGENOME® siRNA reagents, the ON-TARGETPlus™ line of siRNA products have two major differences. The first difference is that ON-TARGETPlus™ reagents are designed to reduce off-target effects with ON-TARGET modification patterns which encourage correct anti-sense uptake and discourage sense-strand interference, a common factor in mild to severe cases of off-target effect. Additionally, Dharmacon has found a way to increase overall siRNA specificity using SMARTpool, effectively utilizing four distinct siRNAs to target one gene.

Ordering is straightforward on the Dharmacon, Inc. website, with a comprehensive procedure to ensure that the designated targeting sequence is the correct sequence, and allows the investigator to select siRNA targeting any human, mouse, or rat gene in RefSeq.

We ordered two selections of siRNA from Dharmacon: ON-TARGETPlus™ siRNA for protein RhoA in rats, and Dharmacon’s Standard siRNA control reagents. Packaging was straightforward, with siRNA arriving in the selected quantity in dry form. Preparing aliquots was error-proof, involving mixing an appropriate volume of media to achieve the desired concentration. The only difficulty stemmed from the invisible nature of the dry siRNA particles- indeed, a small mold mark in the plastic container or a scuff can easily be mistaken as visible siRNA salt. While there was no positive way of visually confirming the homogeneity of the final siRNA solution, the concentration can be checked by a laboratory spectrophotometer. Both the ON-TARGETPlus™ targeting siRNA and the control aliquots showed the expected siRNA concentrations after examination in a spectrophotometer. The samples were flash-frozen in a liquid nitrogen bath for storage and later thawed at room temperature for experimental use.

Incorporation of the siRNA into the experiment also revealed no complications. Transfection of rat neuronal cells was conducted with the help of Invitrogen’s Lipofectamine transfection agent at the concentrations listed under Invitrogen protocols. While there was no way to test successful transfection agent-siRNA interactions and formation of “lipid bubble” complexes, success was measured by the level of expressed proteins in transfected neurons.

Side-by-side use of control and targeting siRNAs yielded acceptable levels of expression suppression, as shown by an ImageXpress instrument measuring stain intensities of the targeted protein across control and transfected siRNA (what appears to be 20-40%). Consistency of suppression was widely varying from experiment to experiment, though this may have been due to other factors, such as cell type and transfection agent. The results were somewhat comparable to those obtained with Ambion’s siRNA reagents, though consistency was less than expected. For the price, Dharmacon’s ON-TARGETPlus™ siRNA reagents remain a good choice for investigators, demonstrating reasonable efficacy and acceptable levels of consistency.