In vitro gene silencing assay

Primary mouse hepatocytes were obtained from Life Technologies and cultured in Williams E Medium with 10% foetal bovine serum. Transfection was carried out by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax (Invitrogen) per well to 5 μl of each siRNA duplex at the desired concentration to an individual well in a 384-well plate. The mixture was incubated at room temperature for 20 min, and 40 μl of complete growth media containing 5000 cells was added to the siRNA mixture. Samples were incubated for 24 h, and then RNA was isolated. A similar procedure was followed for the transfection of 10 000 000 cells and scaled accordingly. Dose response experiments were done using eight 6-fold serial dilutions over the range of 20 nM to 75 pM or 50 nM to 187.5 pM.

RNA was isolated using a Dynabeads mRNA Isolation Kit (Invitrogen). Cells were lysed in 75 μl of Lysis/Binding Buffer containing 3 μl of beads per well and mixed for 10 min on an electrostatic shaker. Buffers were prepared according to the manufacturer's protocol. The washing steps were automated on a Biotek EL406 using a magnetic plate support. Beads were washed once in buffer A, once in buffer B, and twice in buffer E, with 90 μl volume per wash and with aspiration steps between washes.

cDNA synthesis was accomplished with the ABI High-capacity cDNA Reverse Transcription kit (Applied Biosystems). A mixture of 1 μl of 10× buffer, 0.4 μl of 25 × dNTPs, 1 μl of random primers, 0.5 μl of reverse transcriptase, 0.5 μl of RNase inhibitor, and 6.6 μl of water per reaction were added per well. Plates were sealed, agitated for 10 min on an electrostatic shaker, and then incubated at 37°C for 2 h. Following this, the plates were agitated at 80°C for 8 min. cDNA (2 μl) was added to a master mix containing 0.5 μl mouse GAPDH TaqMan Probe (Applied Biosystems, Cat.# 4308313), 0.5 μl of mouse TTR or F12 TaqMan probes (Applied Biosystems), and 5 μl of Lightcycler 480 probe master mix (Roche) per well in a 384-well plate (Roche). Real-time PCR was performed in an ABI 7900HT RT-PCR system (Applied Biosystems) using the ΔΔCt (RQ) assay. Each siRNA concentration was tested in four biological replicates. To calculate relative fold change, real-time data were analysed using the ΔΔCt method and normalised to assays performed with cells transfected with 10 nM control siRNA. IC₅₀ values were calculated using a four-parameter fit model using XLFit.