Lightcycler run 4

AChE, BChE, and BACE-1 mRNAs levels were analyzed by two-step quantitative real-time PCR (qRT-PCR), in a volume of 10 μL reaction mixture, using relative quantification methodology with a LightCycler TM Instrument (Roche, Germany) and a LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science), according to the manufacturer's instructions. All primer sequences were self-designed using Oligo 6.0 software (National Biosciences) and verified by the electrophoretic assessment and by a single temperature dissociation peak (melting curve analysis) of each cDNA amplification product. An GAPDH gene was used as a housekeeping gene (endogenous internal standard). Standard curves were prepared from dilution of cDNA and generated from a minimum of four data points for each quantified gene. All quantitative PCR reactions were repeated twice. Data were evaluated using LightCycler Run 4.5 software (Roche Applied Science). Each PCR run included a nontemplate control to detect potential contamination of reagents.

The acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and beta-secretase (BACE1) genes expression level was analyzed by two-step quantitative real-time PCR (qRT-PCR), in a volume of 10 μL reaction mixture, using relative quantification methodology with a LightCycler TM Instrument (Roche, Germany) and a LightCycler Fast Start DNA Master SYBR Green I kit (Roche Applied Science) according to the instructions of the manufacturer. All primers sequences were designed and custom-designed using the Oligo 6.0 software (National Biosciences) and were verified by assessment of a single PCR product on agarose gel and by a single temperature dissociation peak (melting curve analysis) of each cDNA amplification product. An GAPDH gene was used as a housekeeping gene (endogenous internal standard) for normalization of qPCR. For each quantified gene, standard curves were prepared from dilution of cDNA and generated from a minimum of four data points. All quantitative PCR were repeated twice. The data were evaluated using LightCycler Run 4.5 software (Roche Applied Science). Each PCR run included a nontemplate control to detect potential contamination of reagents.

The genes (VEGF, TGF-β1, TGF-β2, TGF-β3, Muc5AC) expression level was analyzed by a two-step quantitative real-time PCR (qRT-PCR) reaction in the lung and bronchi of rats, in 10 µL of reaction mixture, using relative quantification methodology with a LightCycler TM Instrument (Roche Applied Science, Mannheim, Germany) and a LightCycler Fast Start DNA Master SYBR Green I kit (Roche Applied Science, Mannheim, Germany), according to the instructions of the manufacturer. All primer sequences were designed using the Oligo 6.0 software (National Biosciences, Colorado Springs, CO, USA) and were verified by an assessment of a single PCR product on agarose gel and by a single temperature dissociation peak (melting curve analysis) of each cDNA amplification product (Table 6).
The GAPDH gene was used as a housekeeping gene (endogenous internal standard) for normalization of the qPCR reaction. For each quantified gene, standard curves were prepared from dilution of cDNA. All quantitative PCR reactions were repeated twice. The data was evaluated using Light Cycler Run 4.5 software (Roche Applied Science, Mannheim, Germany). Each PCR run included a non-template control to detect potential contamination of reagents (Table 7).