Nucleospin rna 2 columns

N2a58 and N2a22L were incubated in the presence of CA and CS for 48 h and then total RNA was extracted using Macherey Nagel Nucleospin RNA II columns, following the manufacturer’s protocol. The purified RNA was quantified spectrophotometrically using Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA). cDNA synthesis was performed using TaKaRa PrimeScriptTM RT reagent kit (Protocol: Reverse transcription) starting with 500 ng of total RNA.

Total RNA was extracted from ES cells or tissue samples with NucleoSpin RNA II columns (Macherey-Nagel). ES cells from at least two to three different wells of 24-well plates were always pooled together to compensate for variability in cell seeding. RNA quantity and RNA quality were assessed by gel electrophoresis. For each sample, 200 ng of total RNA was reverse-transcribed (Eurogentech, RT Core kit). Amplified cDNA was quantified using GoTaq qPCR Master Mix (Promega) on a Rotor-Gene 6000 (Corbett). Primers used for amplification which were not previously reported [9 (link)] were taken from PrimerBank [63 (link)]. Amplification take-off values were extracted using the built-in Rotor-Gene 6000 relative quantitation analysis function and relative expression was calculated with the 2^-ΔΔCt method, normalizing to the housekeeping gene β-actin. Standard errors shown as error bars in all histograms were obtained from the error propagation formula as described in [64 (link)]; the statistical significance of three independent experiments was probed with a randomization test using the REST software [65 (link)].

RNA from cultured cells was extracted and purified using Nucleo Spin RNA II columns (Machery-Nagel Inc., Easton, PA USA). First strand cDNA was synthesized from RNA using random hexamer and oligo dT priming and moloney murine leukemia virus reverse transcriptase (MMLV, Life Technologies) to produce random primed cDNA. Quantitative PCR measurements were performed using the SA Bioscience SYBR Green Supermix (QIAGEN, Valencia, CA USA) reagents on an iCycler Real-Time PCR Detection System (Bio-Rad Inc., Hercules, CA USA). Each reaction was carried out in 15 μl reaction volumes of SA Bioscience SYBR Green Supermix with 5 pM of each primer and diluted first-strand cDNA. All cDNA samples were measured in duplicate. Ribosomal protein large protein 38 (Rplp38) was used as the housekeeping gene control for ΔC_T calculation [ΔC_T  =  (C_T of the target gene) – (average C_T of housekeeping genes)]. Primer sequences were listed in Table S2. Fold change expression values were calculated using 2^−ΔΔCT methods[57] (link), where ΔΔC_T  =  (ΔC_T of the treatment sample) – (ΔC_T of control treatment samples) with no added IGF1 as control treatment and normalized to 1.

According to the manufacturer’s instructions, total RNA was extracted using NucleoSpin RNA II columns (Macherey–Nagel, Hœrdt, France). cDNA was synthesized by reverse transcription of 1 μg total RNA with M-MLV Reverse Transcriptase, RNase H Minus, and Point Mutant reverse transcriptase (Promega, Leiden, The Netherlands) using oligo(dT) primers (Promega). cDNAs were used as a template for subsequent quantification by real-time PCR in a reaction mixture containing 1 × Power SYBR® Green PCR Master Mix (Applied Biosystems, Halle, Belgium) and 0.1 μM of each primer (sequences available on request). Amplification was performed on an ABI 7300 real-time PCR system (Applied Biosystems, Lennik, Belgium). The amount of target gene was normalized to the endogenous level of β-actin using the 2^−ΔCT method.

Total RNA from lung tissue was isolated with TRI reagent (Sigma-Aldrich GmbH, Deisenhof, Germany) according to the manufacturer's protocol, followed by purification with NucleoSpin RNA II columns (Macherey&Nagel, Düren, Germany). The complementary deoxyribonucleic acid was synthesized with the Revert AidTM H Minus First Strand Synthesis Kit (MBI Fermentas, St. Leon Roth, Germany) from 1 μg total RNA according to instructions of the fabricant. Using cyclophilin A and ß2-microglobulin as housekeeping genes, the mRNA expression of the inflammatory mediators tumor necrosis factor α (TNF-α), interleukin 6 and 8 (IL-6, IL-8), amphiregulin and tenascin-c were quantified with quantitative real-time polymerase chain reaction (Maxima SYBR Green qPCR MasterMix, Fermentas, St. Leon Roth, Germany) in the iCycler MyiQ2 real time polymerase chain reaction system (BioRad, Munich, Germany). The total protein content in lung tissue was measured using the BioRad Protein Assay (BioRad, Munich, Germany). Protein levels of TNF-α, IL-6, and IL-8 were measured in lung tissue using commercial ELISA kits (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions.

For RT-PCR, total RNA from BCi-NS1 and CFBE human airway epithelial cells was isolated using NucleoSpin RNA II columns (Macherey-Nagel, Düren, Germany). Total RNA (0.5 µg/25 µL reaction) was reverse-transcribed using random primer (Promega, Mannheim, Germany) and M-MLV reverse transcriptase RNase H minus (Promega, Mannheim, Germany). Each RT-PCR reaction contained sense (0.5 µM) and antisense primer (0.5 µM) (Table 1), 0.5 µL cDNA and GoTaq polymerase (Promega, Mannheim, Germany). After 2 min at 95 °C, cDNA was amplified (targets of 35 cycles, reference GAPDH 25 cycles) for 30 s at 95 °C, 30 s at 56 °C and 1 min at 72 °C. PCR products were visualized by loading on Midori Green Xtra (Nippon Genetics Europe) containing agarose.