Prolactin and Brown Adipocytes Figure 6. Proposed model of regulation of brown adipocyte function by PRL/JAK2/STAT5 /IGF-2 pathway doi:10.1371/journal.pone.0001535.g006 as previously described [45]. Table 1 indicates primer sequences of genes analyzed by RT-PCR or by quantitative real time PCR. Quantitative real time PCR Specific gene expression was quantified by real time PCR, using an ABI 7300 Sequence Detector (Applied Biosystems, Foster City, CA). Briefly, 1 mg of total RNA was treated using DNase I Amplification Grade procedure (Invitrogen). RNA was thereafter reverse- transcribed with 200 units of reverse transcriptase using the SuperscriptTM II kit (InVitrogen), then samples were diluted 10- fold and 1/20th of the reverse transcription reaction was used for PCR using the qPCRTM Mastermix Plus for SybrTM Green I (Eurogentec, Seraing, Belgium), as previously described [45]. Controls without reverse transcriptase and without template were included to verify that fluorescence was not overestimated by residual genomic DNA amplification or from primer dimer . .. . Table 1. Primer sequences of genes analyzed in PCR, RT-PCR and Real Time PCR .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... Name Accession numbe Amplicon Sense primer Antisense primer . ... 18S X00686 66 bp CCCTGCCCTTTGTACACACC CGATCCGAGGGCCTCACTA . ... PRLR BC005555 673 bp GAGAAAAACACCTATGAATGTC AGCAGTTCTTCAGACTTGCC .... PPARc2 U09138 161 bp GCATCAGGCTTCCACTATGGA AAGGCACTTCTGAAACCGACA ... SREBP1 BC056922 152 bp CGGCTGTTGTCTACCATAAGCTG CATAGATCTCTGCCAGTGTTGCC . ... PGC-1a AF049330 162 bp CCCAGGCAGTAGATCCTCTTCAA CCTTTCGTGCTCATAGGCTTCATA .... UCP1 BC012701 150 bp TCACCTTCCCGCTGGACACT GCAGGCAGACCGCTGTACAG ... b3AdR NM_013462 150 bp TGCGCACCTTAGGTCTCATTAT AAGGCGGAGTTGGCATAGC . . . .. The abbreviations of the genes, their GENBANK or NCBI accession number, the size of the amplicon and 59 to 39nucleotide sequences of the sense and antisense primers ... are presented. .. doi:10.1371/journal.pone.0001535.t001 PLoS ONE | www.plosone.org 10 February 2008 | Issue 2 | e1535