Taqman assay fluorogenic 5 nuclease chemistry

Duplicate 25-ng cDNA samples were subjected to qPCR analysis using Rotor-Gene 6000 real-time rotary analyzer (Corbett Life Science; Concord NSW, Australia) with TaqMan Assay fluorogenic 5′nuclease chemistry (Invitrogen) as the fluorophore. Final concentrations for a 10-μL qPCR reaction with 25-ng of loaded cDNA include 0.25 U of AmpliTaq Gold DNA polymerase (Roche), 1.25 mM MgCl₂ (Roche), 100 μM dNTPs and 10× PCR buffer (Roche). Briefly, the samples are heated at 95 °C for 10 min. Samples are then subject to being heated at 95 °C for 10 s and then 58 °C for 45 s for a total of 45 cycles. Expression levels were normalized to that of peptidylprolyl isomerase A (PPIA) for tissues or Polr2A for cells mRNA using the delta–delta CT method (2^−ΔΔCT). Gene expression data were gathered from various tissues. Arg 1 (Mm00475988_m1), Basph8 (Mm00484933_m1), Gata3 (Hs00231122_m1), Nos2 (Mm00440502_m1), Polr2a (Mm00839493_m1), Ppia (Mm02342430_g1), Siglecf (Mm00523987_m1), SERT (Mm00439391_m1), Tph1 spanning exons 2-3 (Mm01202614_m1), Tph1 probe spanning exon 4-5 (Mm00493794_m1), Tph2 (Mm00557715_m1), Tpsb2 (Mm01301240_g1), and Ucp1 (Mm01244861_m1) from Lifetechnologies were used.

The RNeasy Mini Kit (QIAGEN: Hilden, Germany) was used for total RNA purification. Samples were reverse transcribed to cDNA using SuperScript IV reverse transcriptase (Invitrogen: Waltham, MA). Amplification and detection were performed in a qPCR thermocycler (Corbett Rotor Gene 6000: Montreal Biotech Inc: Dorval, Canada) using TaqMan Assay fluorogenic 5′ nuclease chemistry (Invitrogen). Relative gene expression was calculated using the Livak method [31 (link)] normalizing to GAPDH. See Table S1 for list of probes.