strain background: BALB/c genotype/variation: wild type treated with: PBS (control) tissue: lung, right lobe
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from right lobes of unsensitized or sensitized lungs using the TRIZOL method (InVitrogen), followed by digestion with RNase-free DNase (Qiagen) and purification on RNeasy spin columns using RNeasy Minikit (Qiagen). In each experiment, for qRT-PCR gene expression array RNA was pooled from 4-5 mice per group.
Label
SYBR Green
Label protocol
Quantitative reverse transcription real-time PCR (qRT-PCR) was used to quantify the amounts of mRNA in the lungs using custom RT2 Profiler PCR Arrays manufactured by Qiagen/SA Biosciences. The arrays included 40 assay genes, 5 housekeeping genes, and reverse transcription efficiency and DNA contamination controls. All primer sets were from Qiagen/SA Biosciences, except for the Pglyrp1 primers. cDNA was synthesized from 2 µg of RNA using RT2 PCR Array First Strand Kit (Qiagen/SA Biosciences) and the arrays were performed according to the manufacturer instructions using Qiagen/SA Biosciences Master Mix. Each experiment was performed on RNA pooled from 4–5 mice/group and repeated 3 times with new groups of mice.
Hybridization protocol
n/a
Scan protocol
n/a
Description
WT PBS-1
Data processing
For each gene, ΔCt was calculated using the same threshold (0.2) for all genes and Ct>35 considered as no expression, followed by normalization to 5 housekeeping genes (Hsp90ab1, Gusb, Hprt1, Gapdh, and Actb) included in each array, followed by calculation of ΔΔCt for each gene from two arrays: ΔΔCt = ΔCt1 – ΔCt2, where ΔCt1 is the HDM-sensitized mice group and ΔCT2 is the unsensitized mice group, using the program provided by Qiagen/SA Biosciences.
