Sybr green fast advanced cells to ct kit

For monocyte and macrophage gene expression analyses, total RNA was extracted with Trizol reagent (Thermo Fisher Scientific), following manufacturer’s instructions. RNA concentration and estimation of purity were determined by absorbance reading at 260 and 280 nm with NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). cDNA was synthesized by reverse transcription using the Advantage RT-for-PCR Kit (Clontech) and used to perform SYBR Green based qPCR analysis. For airway epithelial cells gene expression analysis, cell lysis, total RNA extraction and cDNA synthesis were performed using the SYBR Green Fast Advanced Cells-to-Ct Kit (Thermo Fisher Scientific) following manufacturer’s protocol.
All the qPCR amplifications were achieved by Step One Plus or QuantStudio 12K Flex Real Time PCR systems (Thermo Fisher Scientific). The data were analyzed using the double delta Ct (DDCt) method and the 2^-DDCt values were displayed. The set of primers used are listed in Table 1.

Total RNA was isolated using the Ultraspec system (Biotecx, Houston, TX, USA) according to the manufacturer’s instructions. The cytoplasmic and nuclear RNA was isolated and purified using RNeasy Kits (Qiagen) following the manufacturer’s protocol. Quantitative PCR was performed using SYBR Green Fast Advanced Cells-to-CT Kit (Thermo Fisher) on an ABI PRISM 7900 Sequence Detector System (Applied Biosystems, Foster City, CA, USA). GAPDH or U6 was used as an internal reference. PCR reactions were performed in triplicate. Gene expression was quantified using the 2^−△△ct method. The primer sequences are summarized in Table 2.

To measure RNAi knockdown (Figure S3E), RNA was isolated from HEK293T cells 72 h after the second siRNA transfection and converted to cDNA using the SYBR Green Fast Advanced Cells-to-CT Kit (Thermo Fisher Scientific) with cell lysis for 10-15 min using lysis solution containing 1:50 DNaseI to fully digest genomic DNA. All other steps were carried out according to the manufacturer’s protocol. Each 20 μL qPCR reaction was performed in technical and biological triplicate with 500 nM of each primer, 2 μL cDNA, 1 × SYBR Green (Thermo Fisher Scientific), and 10 μL Q5 High-Fidelity 2 × Master Mix (New England BioLabs) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) with the following thermocycling conditions: 98°C for 2 min, and 40 cycles of [98°C for 15 s, 65°C for 20 s, and 72°C for 30 s]. β-actin (ACTB) served as a housekeeping gene to normalize the amount of cDNA in each qPCR reaction. Relative RNA abundances from gene knockdown were calculated in comparison to a non-targeting siRNA control by the 2^-ΔΔCT method. A list of primers used for qPCR reactions is provided in Table S4.