RNA was prepared from BAL cells using a QIAGEN® QIAshredder™ kit (Venlo, Limburg, Netherlands) and converted to cDNA. The cell samples for each treatment group were pooled into one larger sample for analysis. Thermocycling was used to analyze mRNA expression of CCL2 (mCCL2 1F103: CCT GCT GCT ACT CAT TCA CCA; mCCL2 1R259: GTC TGG ACC CAT TCC TTC TTG), IL1B (mIL1b1F216: TGT GGC AGC TAC CTG TGT CTT; mIL1b1R321: TCC CAT GAG TCA CAG AGG ATG), IL6 (mIL6F23: GAC TTC CAT CCA GTT GCC TTC; mIL6R125: TGG GAG TGG TAT CCT CTG TGA), CXCL10 (mCXCL10F12: AGT GCT GCC GTC ATT TTC TGC; mCXCL10R139: CCT ATG GCC CTC ATT CTC ACT) and IL12B (mIL12bF131: GTG ACA CGC CTG AAG AAG ATG; mIL12bR303: CTT CTT GTG GAG CAG CAG ATG). The ABI 7900HT Fast Real-Time PCR System by Applied Biosystems (Waltham, MA) was used and primers were designed with OligoPerfect™ Designer (lifetechnologies.com, Carlsbad, CA). Fold expression was calculated by the ΔΔCT method normalizing to the water (control) treatment group and using actin as the control gene (Sarkar et al., 2012 (link)).
Protocol detail
Quantification of Inflammatory Gene Expression
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Actin
Ccl2
Cdna
Gene control
Il1b
Mrna
Primers
Samples treatment
Corresponding Organization : Rutgers, The State University of New Jersey
Other organizations : London Centre for Nanotechnology, Imperial College London, University of Malaya, Duke University, University of Southern California
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Variable analysis
independent variables
- Treatment group
- MRNA expression of CCL2
- MRNA expression of IL1B
- MRNA expression of IL6
- MRNA expression of CXCL10
- MRNA expression of IL12B
- Actin (control gene)
- Water (control treatment group)
- Water (control treatment group)
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