Thunderbird sybr green

Quantitative polymerase chain reaction (qPCR) was performed with the cDNA derived in the previous section, THUNDERBIRD® SYBR Green (Toyobo, Osaka, Japan) and the primer sets in Table 2 on Thermal Cycler Dice Real Time System TP850 (TaKaRa, Shiga, Japan). The thermal cycling protocol of qPCR was as follows: initial denaturation at 95 ∘ C for 1 min and followed by 45 cycles of amplification. Each cycle consisted of 95 ∘ C for 10 s and 60 ∘ C for 30 s. The qPCR programme ended with dissociation curve analysis in the following order: 95 ∘ C for 15 s, 60 ∘ C for 30 s and 95 ∘ C for 15 s. The absolute quantity of each gene was derived from the second Derivative Maximum Method. Both the transcription levels of GbDHCR24-1 and GbDHCR24-2 were normalized to the that of β -actin. All datapoints were performed twice to confirm the reproducibility.

Total RNA from mouse cerebellum was purified using the RNeasy mini kit (Qiagen, Limburg, Netherlands). Purified total RNA was reverse-transcribed using SuperScript VILO (Invitrogen). Quantitative PCR analyses were performed on a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using the Thunderbird SYBR Green (Toyobo, Osaka, Japan). The primer sequences for RORα target genes, based on information in a previous report, were as follows^{24 (link)}:
Mouse A2bp1: forward, 5′-AGACCACTGTCCCTGACCAC-3′; reverse, 5′-CATTTGTCGGAGGTCTGGAT-3′.
Mouse Cyp19a1: forward, 5′-CTTTCAGCCTTTTGGCTTTG-3′; reverse, 5′-ATTTCCACAAGGTGCCTGTC-3′.
Mouse Ctgf: forward, 5′-TGCGAAGCTGACCTGGAGGAAA-3′; reverse, 5′- CCGCAGAACTTAGCCCTGTATG-3′.
Mouse Cyr61: forward, 5′-GTGAAGTGCGTCCTTGTGGACA-3′; reverse, 5′- CTTGACACTGGAGCATCCTGCA-3′.
Mouse Ankrd1; forward, 5′-GCTTAGAAGGACACTTGGCGATC-3′; reverse, 5′-GACATCTGCGTTTCCTCCACGA-3′.
Mouse Bcl-2; forward, 5′-CCTGTGGATGACTGAGTACCTG-3′; reverse, 5′- AGCCAGGAGAAATCAAACAGAGG-3′.
Mouse c-Flip; forward, 5′-GCTCTACAGAGTGAGGCGGTTT-3′; reverse, 5′- CACCAATCTCCATCAGCAGGAC-3′.
Mouse XIAP; forward, 5′- GGCAGAATATGAAGCACGGATCG-3′; reverse, 5′- CACTTGGCTTCCAATCCGTGAG-3′.
Mouse Naip1: forward, 5′- CGAGGTCTCAGAGACAAACCAG-3′; reverse, 5′- GAACTCTCCAGGAAGGACTGAG-3′.

Total RNA was purified with NucleoSpin RNAII (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). To eliminate genomic DNA contamination, on-column DNA digestion was carried out for each sample with DNase I. The purified total RNA was reverse-transcribed with SuperScript VILO (Invitrogen, Carlsbad, CA, USA). Quantitative PCR analyses were performed with the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the Thunderbird SYBR Green (TOYOBO, Osaka, Japan). The primer sequences were; mThy1_3F: CAGCAACTGGAGGCGTTGG for a mouse thy-1 promoter and hAPP_56R: TCCCACTCGCACAGCAGC for a 5′ untranslated region of human APP in a transgenes; rGAPDH_2L: 5′-AGCCCAGAACATCATCCCTG-3′ and rGAPDH_2R: 5′-CACCACCTTCTTGATGTCATC-3′ for mouse GAPDH.