Rt enzyme mix

One day prior to free uptake assays cells were seeded into 96-well plates (∼10,000 cells per well) and allowed to attach for at least 16 hours. ASOs were diluted into complete growth over a 12-point 3-fold dilution series at 10× final concentration. Diluted ASOs were then applied to triplicate treatment wells at 0.1× final volume and plates were returned to the incubator for 20–24. Total RNA was prepared using an RNeasy mini kit (Qiagen, Valencia, CA, USA) from cells grown in 96-well plates. qRT-PCR was performed using TaqMan primer probe sets. Briefly, ∼50 ng total RNA in 5 μl water was mixed with 0.3 μl primer probe sets containing forward and reverse primers (10 μM of each) and fluorescently labeled probe (3 μM), 0.3 μl RT enzyme mix (Qiagen), 4.4 μl RNase-free water, and 10 μl of 2 × PCR reaction buffer in a 20 μl reaction. Reverse transcription was performed at 48°C for 10 min, 40 cycles of PCR were conducted at 94°C for 20 s, and 60°C for 20 s within each cycle, using StepOne Plus RT-PCR system (Applied Biosystems, Phoenix, AZ, USA). The mRNA levels were normalized to the amount of total RNA present in each reaction as determined for duplicate RNA samples by Ribogreen assay (Life Technologies).

Total RNA was prepared using an RNeasy mini kit (QIAGEN, Valencia, CA, USA) from cells grown in 96-well plates (around 10,000 cells per well). qRT-PCR using TaqMan primer probe sets was performed essentially as described previously.³³ (link) In brief, approximately 50 ng total RNA in 5 μL water was mixed with 0.3 μL primer probe sets containing forward and reverse primers (10 μM of each) and fluorescently labeled probe (3 μM), 0.3 μL RT enzyme mix (QIAGEN), 4.4 μL RNase-free water, and 10 μL of 2× PCR reaction buffer in a 20 μL reaction. Reverse transcription was performed at 48°C for 10 min; 40 cycles of PCR were conducted at 94°C for 20 s and 60°C for 20 s using the StepOne Plus RT-PCR system (Applied Biosystems). Levels of mRNA were normalized to the amount of total RNA present in each reaction as determined for duplicate RNA samples using the RiboGreen assay (Life Technologies).

Total RNA was prepared using an RNeasy mini kit (Qiagen, Valencia, CA, USA) from cells grown in 96-well plates (∼10 000 cells per well). qRT-PCR using TaqMan primer probe sets were performed essentially as described previously (15 (link)). Briefly, ∼50 ng total RNA in 5 μl water was mixed with 0.3 μl primer probe sets containing forward and reverse primers (10 μM of each) and fluorescently labeled probe (3 μM), 0.3 μl RT enzyme mix (Qiagen), 4.4 μl RNase-free water, and 10 μl of 2× PCR reaction buffer in a 20 μl reaction. Reverse transcription was performed at 48°C for 10 min, 40 cycles of PCR were conducted at 94°C for 20 s, and 60°C for 20 s within each cycle, using StepOne Plus RT-PCR system (Applied Biosystems, Phoenix, AZ, USA). The mRNA levels were normalized to the amount of total RNA present in each reaction as determined for duplicate RNA samples by Ribogreen assay (Life Technologies).

Total RNA was prepared using an RNeasy mini kit (Qiagen, Valencia, CA, USA) from cells grown in 96-well plates (around 10 000 cells per well). qRT-PCR using TaqMan primer probe sets were performed essentially as described previously (16 (link)). Briefly, ∼50 ng total RNA in 5 μl water was mixed with 0.3 μl primer probe sets containing forward and reverse primers (10 μM of each) and fluorescently labeled probe (3 μM), 0.3 μl RT enzyme mix (Qiagen), 4.4 μl RNase-free water, and 10 μl of 2× PCR reaction buffer in a 20 μl reaction. Reverse transcription was performed at 48°C for 10 min, 40 cycles of PCR were conducted at 94°C for 20 s and 60°C for 20 s using the StepOne Plus RT-PCR system (Applied Biosystems, Phoenix, AZ, USA). The mRNA levels were normalized to the amount of total RNA present in each reaction as determined for duplicate RNA samples by Ribogreen assay (Life Technologies).

Gene expression values were calculated using the comparative delta delta C_t (–ΔΔC_t) method and expression against pooled RNA standard curve, using housekeeper gene C_t values and control-treated C_t for normalization as calculated on StepOne Analysis Software v2.3. Total RNA was prepared using an RNeasy mini kit (Qiagen) from cells grown in 96-well plates (*10 000 cells per well). qRT-PCR using TaqMan primer probe sets were performed as described previously (28 (link)). In brief, 50 ng total RNA in 5 μl water was mixed with 0.3 μl forward and reverse primers (10 μM of each) and fluorescently labeled probe (3 μM), 0.3 μl RT enzyme mix (Qiagen), 4.4 μl RNase-free water, and 10 μl of 2 · PCR reaction buffer in a 20 μl reaction. Reverse transcription was performed at 48°C for 10 min; 40 cycles of PCR were conducted at 94°C for 20 s and 60°C for 20 s using the StepOne Plus RT-PCR system (Applied Biosystems). Levels of messenger RNA were normalized to the amount of total RNA present in each reaction as determined for duplicate RNA samples using the RiboGreen assay (Life Technologies) or normalized to Cyclophilin A house-keeping gene expression.

Total RNA was prepared using a RNeasy mini kit (Qiagen) from cells grown in 96-well plates based on the manufacturer's protocol. qRT-PCR was performed in triplicate using TaqMan primer probe sets as described previously (24 (link)). Briefly, approximately 50 ng total RNA in 5 μl water was mixed with 0.3 μl primer probe sets containing forward and reverse primers (10 μM of each) and fluorescently labeled probe (3 μM), 0.5 μl RT enzyme mix (Qiagen), 4.2 μl RNase-free water, and 10 μl of 2× PCR reaction buffer in a 20 μl reaction. Reverse transcription was performed at 48°C for 10 min, followed by 94°C for 10 min, and then 40 cycles of PCR were conducted at 94°C for 30 s, and 60°C for 30 s within each cycle using the StepOne Plus RT-PCR system (Applied Biosystems). The mRNA levels were normalized to the amount of total RNA present in each reaction as determined for duplicate RNA samples using the Ribogreen assay (Life Technologies). Statistical analyses were performed from three independent experiments using Prism with either t-test or F-test for curve comparison based on non-linear regression (dose-response curves) for XY analyses, using equation ‘log (agonist) versus normalized response – variable slope’. The Y axis (relative level) was used as the normalized response.

Total RNA was prepared using an RNeasy Mini Kit (QIAGEN) from cells grown in 96-well plates using the manufacturer’s protocol. qRT-PCR was performed in triplicate using TaqMan primer probe sets as described previously.³³ (link) Briefly, ∼50 ng total RNA in 5 μL water was mixed with 0.3 μL primer probe sets containing forward and reverse primers (10 μM of each) and fluorescently labeled probe (3 μM), 0.5 μL RT enzyme mix (QIAGEN), 4.2 μL RNase-free water, and 10 μL of 2× PCR buffer in a 20-μL reaction. Reverse transcription was performed at 48°C for 10 min, followed by 94°C for 10 min, and then 40 cycles of PCR were conducted at 94°C for 30 s and 60°C for 30 s within each cycle using the StepOne Plus RT-PCR system (Applied Biosystems). The results were analyzed by the relative quantity ddCt (dela-delta Ct) method. The mRNA levels were normalized to the amount of total RNA present in each reaction as determined for duplicate RNA samples using the Ribogreen assay (Life Technologies).