Other tissues were measured as individual samples. kinase inhibitor Crenolanib Real time PCR cycler conditions were 50 C for 2 minutes. 95 C for 10 minutes. and 40 cycles of 95 C for 15 seconds and 60 C for 1 minute. 5 rapid amplification of cDNA ends To determine the transcriptional start sites of Cyp19a1 transcripts from various tissues, 5 RACE was performed Inhibitors,Modulators,Libraries using the SMART RACE cDNA amplification kit. One microgram of total RNA from each tissue underwent RT with a modified oligo primer. After Inhibitors,Modulators,Libraries PowerScript RT reached the end of the mRNA template, it added several dC residues. The SMART II A oligonucleotide annealed to the tail of the cDNA and served as an extended template for PowerScript RT. The primary touchdown PCR reverse primer, binding to exon 3 and 4 coding sequence, was combined with the universal primer A mix provided with the kit.

If the pri mary PCR reaction failed to give the distinct bands of interest, a secondary, or nested PCR reaction was per formed with the reverse nested primer, binding to exon 2 coding region, combined with the nested universal primer A. RACE products were cloned into the pCR TOPO TA cloning Inhibitors,Modulators,Libraries vector and subsequently sequenced. Exon specific RT PCR and real time RT PCR amplification Total RNA was extracted from various mouse tissues and treated with the TURBO DNase following the standard protocol. The treated RNA was then reverse transcribed using oligo primers with Inhibitors,Modulators,Libraries super script III first strand RT kit according to the instructions of the manufacturer.

Inhibitors,Modulators,Libraries To amplify the transcript variants in male gonadal adipose tissue, ovary, testis, and brain, the sequence information of RACE products was used to design forward primers that selleck chemical bound the 5 untranslated regions of these transcripts. The reverse primers were located in coding exon II. The following primer pairs were used Real time PCR cycler conditions were 50 C for 2 minutes. 95 C for 10 minutes. and 40 cycles of 95 C for 15 sec onds and 60 C for 1 minute. Plasmids, transfections, and luciferase assays The potential promoter regions of an adipose specific transcript were amplified by PCR. The primers were introduced at the restriction enzyme KpnI and BglII sites. The PCR fragments were then digested and subcloned into a pGL4. 10 luciferase vector. Briefly, the promoter region was cloned into the location between a synthetic poly signal transcriptional pause site and the luc2 gene of the pGL4. 10 vector. All constructs were reconfirmed by sequencing. The primers amplifying the promoter region were Mouse 3T3 L1 cells were transfected with each of the pro moter constructs using Lipofectamine 2000 transfection reagent according to the manufacturers pro tocol. Reporter plasmid and a pGL4. 74 internal control were transfected per well of 6 well plate for 24 hours.