Nucleospin rna kit

RNA was isolated from the cells using the Nucleospin RNA kit (Clontech). cDNA was made using the PrimeScript first strand cDNA synthesis kit (Clontech). An equal amount of cDNA was amplified with the iQSybr Green Mix (Biorad) using gene specific primers. See quantification and statistical analysis section for details on data analysis.

mRNA was isolated from triplicate samples using either manufacturer’s protocol of the illustra RNAspin Mini Kit (GE Healthcare^#25-0500-72) or NucleoSpin RNA Kit (Takara Clontech^#740955.250). 250–500 ng of mRNA were converted into cDNA using 1X iScript (BioRad^#1708891) manufacturer’s protocol. Samples were assessed by qRT-PCR using SsoAdvanced™ Universal SYBR® Green Supermix (BioRad^#1725274) using the primers listed in Supplementary Table 3 All qRT-PCRs were performed in two to three independent experiments with data normalized to the housekeeping gene RPLP0.

RNA was collected and purified using a NucleoSpin RNA kit (Clontech, Mountain View, CA) and RNA quantity and quality was measured with a spectrophotometer (NanoDrop Inc., Wilmington, DE). Reverse transcription was performed using High Capacity Reverse Transcription (Applied Biosciences, Carlsbad, CA), following manufacturers instructions. Real-time PCR was performed using Sensimax II Hi-ROX kit (Bioline, Taunton, MA) according to manufacturers instructions and run on a 7900HT thermal cycler (Applied Biosystems). The threshold cycle (Ct) was obtained from duplicate samples and averaged. The ΔCt was the difference between the average Ct for the target gene and the housekeeping gene, Hprt. The ΔΔCt was the average ΔCt for a given sample point minus the average ΔCt of control samples. The data are expressed as relative quantification calculated as 2^−ΔΔCt.

At 24 hours after the final challenge, mice were euthanized by i.p. lethal injection of 1 ml of 2.5% Avertin in PBS. Bronchoalveolar lavage (BAL) fluid was collected by intratracheal insertion of a catheter and four lavages of 1 ml of PBS. Lungs were perfused with 5 ml PBS; the right upper lobe was removed and frozen in RNAlater (Thermo Fisher Scientific) for RNA isolation. The left lung was excised, digested with Liberase TM (Roche), and single-cell suspensions obtained. The remaining lung tissue was removed and placed in 10% neutral buffered formalin (Leica Biosystems) for histological analysis. BAL TNF-α was detected by ELISA (R&D Systems). Lung tissue and BAL cellular differential counts were determined by flow cytometry analysis. For quantitative RT-PCR, RNA was isolated with the NucleoSpin RNA kit (Clontech). cDNA was synthesized using PrimeScript Reverse Transcriptase (Takara) and mRNA expression levels assessed using the SYBR Premix Ex Taq II (Takara) according to the manufacturer's instructions. The level of mRNA was normalized to Gapdh expression, and the results were analyzed by the 2^−ΔΔCt method.