Here we compare the effect of different transfection reagents on the insertion and localization of ssDNA-functionalized gold nanoparticles targeted at metallothionein-IIA, in HeLa cells. Metallothioneins are a family of ubiquitous low molecular weight proteins that have well-established regulatory roles in the cell, making them a target of choice for applications in metal ion homeostasis, in the detoxification of heavy metals and as protective cellular stress proteins. They also play an integral role in cell survival via their interaction with the transcription factors NF-kB and p53; and elevated metallothionein levels have been found in a number of cancers. MT-IIA accounts for approximately 50% of the total cellular metallothionein protein, and is induced by a wide variety of environmental stresses and agents. Here, we used cadmium to manipulate the levels of the MT-IIA in the cell, to calibrate gene silencing effects. We now describe a method to quantify the gene silencing properties of small interfering RNA, single-stranded RNA and ssDNA, both free and bound to nanoparticles. We show that ssDNA-dependent knockdown of gene expression using functionalized gold nanoparticles occurs at the mRNA level and results in the loss of the specific mRNA transcript as well as the target protein. This approach to gene knockdown is particularly attractive as it bypasses both translation and post-translational mechanisms. In addition, by targeting mRNA, it also avoids the risk of non-specific gene knockdown. Our study also clearly demonstrates the importance of appropriate reagent selection and validation. In testing three different transfection reagents, we observed varying transfection efficiencies but more importantly, we detected biological artefacts present in HeLa cells that were treated with Lipofectamine 2000 or Matra but not in those that had been treated with GeneJuice. In order to study the transfection efficiency of different transfection reagents, we transfected FITC-tagged ssDNA-functionalized nanoparticles into HeLa cells with a panel of three transfection reagents with different formulations, as follows. Matra is a magnet-assisted nanoparticle-based transfection method for intracellular delivery of nucleic acid, while Lipofectamine 2000 is a cationic liposome-based reagent and GeneJuice consists of a non-toxic cellular protein and a small amount of a proprietary polyamine. At first, and most importantly, we observed that the phasecontrast images of live HeLa cells showed that cells transfected with nanoparticles were largely adherent, with shapes that conformed to those of healthy HeLa cells for all the payloads considered, i.e. unfunctionalised nanoparticles, coated with sequences that were either not specifically targeted to a mammalian transcript or with sequences that targeted the human MT-IIa transcripts.