PCR template method for dsRNA Synthesis | McManus …

Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms.

siRNA Transfection - Transfection Protocol

For transfection of eukaryotic cells with siRNA and miRNA ..

Synthesis of siRNA for RNAi in Drosophila - CSH …

The EGFP stable transfected cell line MDA-MB-231-EGFP cells were used for these studies. Briefly, the cells were grown at 37 °C in humidified atmosphere in L15 medium supplemented with 1% antibiotics, 1% NEAA and 10 % FBS. inhibition on EGFP expression was evaluated by treating the cells with formulations containing PBS, negative control siRNA, naked siRNA, lipofecter/siRNA complex, LMWP/siRNA physical mixture, the bio-reducible Conj-1, or the non-reducible Conj-2). The concentration of siRNA was maintained at 50 nM in all of these formulations. The treated cells were incubated for 12h, followed by the replacement with fresh culture media and further incubation for up to 3 days at 37 °C. The siRNA gene silencing down effect were assessed by the inhibition on EGFP expression, using different methodologies including confocal microscopy, FACS, fluorescence intensity and Western-blot analysis. Experimental details were described in each of the following sections.

Protocol Synthesis of siRNA for RNAi in Drosophila

MDA-MB-231-EGFP cells were seeded at 2×104 cells per well on a glass cover slip for 24 h. After transfection using the aforementioned procedure, cells were rinsed with PBS, fixed with 4% paraformaldehyde solution for 30min at room temperature, and then washed three times with PBS. The media was replaced with 2 mM DAPI (Sigma, USA) Hanks balanced salt solution to stain the nuclei for 10 min, and the cover slip was then placed on a sticky microscopy slide in the presence of the antifade mounting medium (Genemed, USA). The cells were imaged immediately using the Olympus FV-300 laser scanning confocal microscope which was equipped with the FLUOVIEW software (Olympus, Tokyo, Japan).

For example, chemically synthesized or in vitro transcribed siRNAs can be transfected into cells, injected into mice, or introduced into plants.

siRNA Synthesis, synthetic oligos, RNA interference

Human breast cancer cell line MDA-MB-231 cells were utilized for these studies. Briefly, cells were grown at 37 °C in humidified atmosphere in L15 medium supplemented with 1% antibiotics, 1% NEAA and 10 % FBS, according to a previously established protocol []. Formulations containing FITC-labeled siRNA (including: PBS, native siRNA, Lipofecter/siRNA complex, LMWP-PEG/siRNA physical mixture, LMWP/siRNA physical mixture, the LMWP-PEG-S-S-siRNA (Conj-1) and LMWP-PEG-mal-S-siRNA (Conj-2) were then incubated the MDA-MB-231 cells with at 37 °C for 2 h. Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, treated with the DAPI (2μg/ml) nucleic acid staining, and then subjected to confocal laser scanning microscopy analysis using an Olympus FV-300 laser scanning microscope operated with FLUOVIEW software (Olympus, Tokyo, Japan).

Custom siRNA Synthesis | Dharmacon

The proposed conjugation method was illustrated in the Supplementary Section (Fig. S1). In brief, the strategy was based on the unique and unparalleled dynamic movement of the PEG polymer and its yielded shielding effect on the charged molecules to override all of the aforementioned obstacles [, ]. As noted, the hydrated state of the PEG chain was demonstrated to possess an unparalleled spatial dynamic mobility thereby enhancing the solubility of its linked compound and diminishing it from aggregation and detection by the host immune system []. In agreement with these reported observations, our results showed that while strong charge-induced aggregation occurred when unmodified LMWP was mixed with siRNA (the turbid solution; Fig. A.1). In sharp contrast, the reaction mixture remained transparent when either the pegylated LMWP was physically mixed with siRNA (Fig. A.2) or following a successful synthesis of the final LMWP-PEG-S-S-siRNA chemical conjugates (Fig. A.3). These findings provide sound evidence to confirm our hypothesis: i.e. the PEG chain on LMWP-PEG would shield LMWP from charge-induced complexation with siRNA during the coupling process (Fig. A.2), and the extraordinarily dynamic spatial movement of PEG would also prevent the intra- or inter-molecular self-assembly event in the final LMWP-PEG-siRNA conjugates (Fig. A.3). Since both reactants involved in the coupling process contain only a single reactive -SH group, formation of 1:1 (siRNA: LMWP) covalently linked conjugates is therefore warranted. Analysis of the particle size of the reaction mixtures also offered strong support to the above findings. As shown in Fig. B.2, the average particle size of the mixture of native LMWP and siRNA was in the range between 300 - 400 nm, explicitly suggesting the formation of aggregated particles due to charge complexation. In sharp contrast, particle sizes of the physical mixture of LMWP-PEG and siRNA (Fig. B.3) as well as the final LMWP-PEG-S-S-siRNA conjugates (Fig. B.4) were almost non-detectable and similar to that of the ultra-pure water (Fig. B.1), reflecting masking of the electrostatic interaction between the two oppositely charged LMWP and siRNA by PEG. These results further confirmed that the final LMWP-PEG-S-S-siRNA conjugates existed in a hydrated and soluble state.

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The expected average in-house turnover time for siRNA is 5-10 working days. Please note that we perform strict quality controls on each and every oligo. In case one or more oligos do not pass our quality control, they will need to be resynthesized. This may, of course, result in a delay.