Supersciptii

Total bacterial RNA was isolated from bacteria grown under different growth conditions after killing by the addition of 0.2 volumes of 95% ethanol, 5% phenol, pH 4.3. Pellets were resuspended in 10 mM Tris, 1 mM EDTA containing 2 mg ml-1 lysozyme and incubated at 37°C for 30 min. Cell lysis solution (Qiagen, Hilden, Germany) was added and the mixture was incubated at 65°C for 5 min and at room temperature for 10 min. After the addition of precipitation solution (Qiagen, Hilden, Germany) and incubation on ice for 5 min cell debris, proteins and DNA were pelleted. The RNA containing supernatant was mixed with ethanol and loaded on a spin column (Promega). Further RNA purification and DNase digestion was done as described by the manufacturer.
A total of 50 μg of RNA of six separate experiments was reverse transcribed to cDNA and labelled with Cy3- or Cy5-conjugated dCTP (GE Healthcare) using reverse transcriptase (SupersciptII, Invitrogen) and random hexamers as primers. RNA was removed by hot-alkali treatment. Labelled cDNA was purified using a Qiaquick PCR purification kit and quantified by Nano-Drop analysis (ND-1000 Spectrophotometer, Peqlab).

Real-time RT-PCR was carried out as described previously^{16 (link)}. Briefly, Total RNA was isolated from frozen preoptic tissue samples, or lysated primer cultures. The concentration of RNA was adjusted to 2 µg/µL, and it was treated with Amplification Grade DNase I (Invitrogen). Then, cDNA was synthesized using SupersciptII (Invitrogen) as suggested in the kit protocol. The cDNA was subsequently diluted (10x), and 2.5 µL of the resulting cDNA was used as template in PCR reactions using SYBR Green dye (Sigma, St Louis, MO, USA). The PCR reactions were performed with iTaq DNA polymerase (Bio-Rad Laboratories, Hercules, CA, USA) and GAPDH was used as housekeeping gene. The primers were: ACAGCCAGCGCTACAAAGTT and GCGGTATCTACTGGCTCTGC for IGFBP-3, and GCTACCGAGAGGACAGCATC and GCACCATAAGCCTTCAGCTC for TH, and TGCCACTCAGAAGACTGTGG and GTCCTCAGTGTAGCCCAGGA for GAPDH. Cycle threshold (Ct) values were obtained from the linear region of baseline adjusted amplification curves. The GAPDH related values were calculated using the following formula: log(Ct_(GAPDH) − Ct_{(IGFBP3 or TH)}). Statistical analyses were performed by unpaired t-test for comparisons of the two different groups.

RT-PCR was performed using SupersciptII (Invitrogen) as described before [50 (link)]. The applied primers were: CCTTACAAGAATGCCGCTCG and TCTCCAGCGGAAAACAGAGC for Ndufs 5, TGTCATCACAACAGGGAGCC and CATGGGGCAGTGTCTTGACT for Nwd1, AGAGCTATGTCGCCCTATGC and GGGGACTTGAGGCATGGATC for Ecm2, GCTGACTGGCCAATGATCCT and AGACACTGCAAACCACTCCC for LOC103694869, TTGTTGTCAAGGACCGGGAG and TCTCTAGACCGCCCATACCC for Rbm3, CTTGGATGGAGCGGACACAT and AGGAGCTGAACTGTTCTGGC for Mepce, AGGGCAATAACCACCAAGCA and GGGCTGTCGTGGCATATTTG for Gpr34 and TGCCACTCAGAAGACTGTGG and GTCCTCAGTGTAGCCCAGGA for GAPDH.