Rt2 profiler pcr array mousechemokines and receptors kit

IL-33^−/− mice were instilled i.t. with PBS
(n = 4) or 2.5 μg of recombinant IL-33
(rIL-33) (n = 5) in a total of 50 μl PBS. The
lungs were harvested 6 h later, minced, and homogenized in RLT buffer using a
Qiagen TissueRuptor. RNA was purified from each mouse lung homogenate (Qiagen
RNeasy Plus Mini Kit) and cDNA was prepared for each sample (Qiagen
RT² First Strand Kit). RT² Profiler PCR Array Mouse
Chemokines and Receptors kit (Qiagen) was used on pooled samples for
quantitative PCR (qPCR) array, which was performed on a CFX96 Real Time System
(Bio-Rad). Array data were analyzed using the data analysis web portal at
http://www.qiagen.com/geneglobe. Cycle threshold (C_T)
values were normalized to the housekeeping genes Gusb and
Hsp90ab1, after which ΔΔC_Tcalculations were performed and transcript fold change over PBS controls was
determined using the 2^−ΔΔC_Tformula. Results for Ccl2, Ccl7, and
Ccl22 were confirmed by individual qPCRs performed on cDNA
from individual mice. PCR primers were as follows: HPRT, forward
5′-TGATCAGT-CAACGGGGGACA-3′, reverse
5′-TTCGAGAGGTCCTTTTCA-CCA-3′; CCL2, forward
5′-GGCCTGCTGTTCACAGTTGC-3′, reverse
5′-CCTGCTGCTGGTGATCCTCT-3′; CCL7, forward
5′-TGTGCCTGCTGCTCATAGCC-3′, reverse
5′-ACATAGCAGCATGTGGATGCATTG-3′; CCL22, forward
5′-CGCAAGCCTG-GCGTTGTTT-3′, reverse
5′-CCTCCCTGGACCACACCAGA-3′.