Taqman pcr master mix with gene specific primers

Ear (half an ear pinna/mouse) and liver (caudate) were homogenized on a TissueLyser II in Buffer RLT (Qiagen). Total RNA was isolated using Qiagen’s RNeasy mini spin column kits with DNase treatment on a QIAcube automated RNA isolation machine. RNA concentrations and purity were analyzed on a NanoDrop spectrophotometer (Thermo Fisher Scientific). The cDNA (1–2 μg) was prepared on an Eppendorf Mastercycler using Applied Biosystems’ High Capacity Reverse Transcription kit. The cDNA was used on the real-time RT² Profiler PCR Array Mouse peroxisome proliferator-activated receptor (PPAR) Targets and Hepatoxicity (QIAGEN; Product #: PAMM-149ZC and PAMM-093ZC) (see Supp. Table 5) and also used as template for real-time PCR reactions containing TaqMan PCR Master Mix with gene-specific primers (Applied Biosystems) on a 7500 Real-Time PCR System. Relative fold gene expression changes (2^‒ΔΔCT) were determined compared to vehicle controls and normalized for expression of reference gene beta-actin (Taqman) or β2-microtubulin (PPAR and Hepatoxicity arrays). Genes that were evaluated are in Supp. Table 2.

Ears (1 per mouse) or LN were mechanically disrupted on a TissueLyser II in Buffer RLT (Qiagen). Qiazol (ears) or chloroform (LN and EpiDerm) was added to the tissue homogenates, then vortexed and centrifuged for 10min at 12000×g (4°C). Total RNA was extracted from the aqueous phase using Qiagen’s RNeasy mini spin column kits with DNase treatment on a QIAcube work station. RNA concentrations and purity were analyzed on a NanoDrop spectrophotometer (Thermo Scientific). cDNA was prepared on an Eppendorf Mastercycler using Applied Biosystems’ High Capacity Reverse Transcription Kit. The cDNA was used as template for real-time PCR reactions containing TaqMan PCR Master Mix with gene-specific primers (Applied Biosystems) on a 7500 Real-Time PCR System. Relative fold changes in gene expression (2^−ΔΔCT) was determined compared with acetone controls unless otherwise indicated and normalized for expression of housekeeping gene Actb (mouse) or GAPDH (human).

Ear (half an ear pinna/mouse) and liver (caudate) were homogenized on a TissueLyser II in Buffer RLT (Qiagen). Total RNA was isolated using Qiagen’s RNeasy mini spin column kits with DNase treatment on a QIAcube automated RNA isolation machine. RNA concentrations and purity were analyzed on a NanoDrop spectrophotometer (Thermo Fisher Scientific). The cDNA (1–2 μg) was prepared on an Eppendorf Mastercycler using Applied Biosystems’ High Capacity Reverse Transcription kit. The cDNA was used as a template for real-time PCR reactions containing TaqMan PCR Master Mix with gene-specific primers (Applied Biosystems) on a 7500 Real-Time PCR System. Relative fold gene expression changes (2^−ΔΔCT) were determined compared to vehicle controls and normalized for expression of reference gene β-actin (Taqman). Genes that were evaluated are identified in Supplemental Table 2.