Ortalized SCN cell lines generated from mPer2Luc knockin and from

Ortalized SCN cell lines generated from mPer2Luc knockin and from mice with targeted disruption of Per1 and Per2 (Per1ldc/Per2ldc) were used to profile the temporal pattern of miR-142-3p expression. These cell lines were maintained and propagated as described previously [29]. Briefly, cells were grown on laminin-coated 60 mm dishes (Corning, Inc.) in Minimal Essential Ornipressin medium (MEM; Invitrogen, Grand Island, NY) containing 10 Fetal Bovine Serum (FBS), 3000 mg/ml glucose and 292 mg/ml L-glutamine. Fresh medium was applied every 48 h and cultures were split 1:3 to 1:5 every 3? 10457188 days. Prior to experimentation, cells were expanded onto laminin-coated 6-well plates (BD Biosciences, San Jose, CA). Approximately 24 h after plating, the medium was changed so as to reduce the FBS concentration to 5 and on the following day cells were rinsed with calcium-magnesium free (CMF) phosphate-buffered saline and then cultured in serum-free Neurobasal medium containing B27 supplement (1X, Invitrogen). Cultures (n = 5) were harvested at 4 h intervals for 36 h by trypsinization and cell pellets were flash frozen in liquid nitrogen. All samples were stored at 280uC until subsequent analysis of miRNA or mRNA content. RNA extraction and Real-time PCR. Total cellular RNA was later extracted from individual mouse SCN tissue samples and cultures of mPer2Luc and Per1ldc/Per2ldc SCN cells using miRNeasy kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s protocols. Total RNA was estimated using a Nanodrop ND2000 (Thermo Scientific, Rockford, IL). Quantitative real-time PCR analysis for miR-142-3p was conducted using Taqman microRNAassays (Applied Biosystems) as described previously [26]. Briefly, miR-142-3p from individual samples was GNF-7 biological activity reverse transcribed using Taqman MicroRNA Reverse Transcription Kit and the cDNA equivalent of 1.5 ng of total RNA was PCR amplified in an ABI PRISM 7500 Fast sequence detection system using the following standard conditions: 1) heating at 95uC for 10 min, and 2) amplification over 40 cycles at 95uC for 15 sec and 60uC for 1 min. As an endogenous control for differences in sample RNA content and reverse-transcription efficiencies, U6 snRNA was also amplified from the same samples using identical parameters. Using the comparative CT method described in the ABI Prism 7700 Sequence Detection System User Bulletin #2 (PE-ABI), the relative abundance of miR-142-3p was calculated by normalization first to corresponding U6 snRNA levels in each sample and then to a calibrator consisting of pooled cDNA from multiple samples over the entire time series. Relative quantification of Bmal1 mRNA abundance in all samples was performed using SYBR-Green real-time PCR technology (ABI) as described previously [30,31]. To generate single-strand cDNAs, total RNA (1 mg) from individual samples was reverse transcribed using random hexamers and Superscript III reverse transcriptase Kit (Invitrogen). Real-time PCR analysis was performed on duplicate aliquots using the cDNA equivalent of 1 ng of total RNA for each sample. The PCR cycling conditions were: 1) serial heating at 50uC for 2 min and 95uC for 10 min, 2) amplification over 40 cycles at 95uC for 15 sec and 60uC for 1 min, and 3) dissociation at 95uC for 15 sec, 60uC for 1 min, 95uC for 15 sec and 60uC for 15 sec. To control for differences in sample RNA content, cyclophilin A (Ppia) was amplified with the cDNA equivalent of 1 ng total RNA from the same samples. Consistent with our previous st.Ortalized SCN cell lines generated from mPer2Luc knockin and from mice with targeted disruption of Per1 and Per2 (Per1ldc/Per2ldc) were used to profile the temporal pattern of miR-142-3p expression. These cell lines were maintained and propagated as described previously [29]. Briefly, cells were grown on laminin-coated 60 mm dishes (Corning, Inc.) in Minimal Essential Medium (MEM; Invitrogen, Grand Island, NY) containing 10 Fetal Bovine Serum (FBS), 3000 mg/ml glucose and 292 mg/ml L-glutamine. Fresh medium was applied every 48 h and cultures were split 1:3 to 1:5 every 3? 10457188 days. Prior to experimentation, cells were expanded onto laminin-coated 6-well plates (BD Biosciences, San Jose, CA). Approximately 24 h after plating, the medium was changed so as to reduce the FBS concentration to 5 and on the following day cells were rinsed with calcium-magnesium free (CMF) phosphate-buffered saline and then cultured in serum-free Neurobasal medium containing B27 supplement (1X, Invitrogen). Cultures (n = 5) were harvested at 4 h intervals for 36 h by trypsinization and cell pellets were flash frozen in liquid nitrogen. All samples were stored at 280uC until subsequent analysis of miRNA or mRNA content. RNA extraction and Real-time PCR. Total cellular RNA was later extracted from individual mouse SCN tissue samples and cultures of mPer2Luc and Per1ldc/Per2ldc SCN cells using miRNeasy kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s protocols. Total RNA was estimated using a Nanodrop ND2000 (Thermo Scientific, Rockford, IL). Quantitative real-time PCR analysis for miR-142-3p was conducted using Taqman microRNAassays (Applied Biosystems) as described previously [26]. Briefly, miR-142-3p from individual samples was reverse transcribed using Taqman MicroRNA Reverse Transcription Kit and the cDNA equivalent of 1.5 ng of total RNA was PCR amplified in an ABI PRISM 7500 Fast sequence detection system using the following standard conditions: 1) heating at 95uC for 10 min, and 2) amplification over 40 cycles at 95uC for 15 sec and 60uC for 1 min. As an endogenous control for differences in sample RNA content and reverse-transcription efficiencies, U6 snRNA was also amplified from the same samples using identical parameters. Using the comparative CT method described in the ABI Prism 7700 Sequence Detection System User Bulletin #2 (PE-ABI), the relative abundance of miR-142-3p was calculated by normalization first to corresponding U6 snRNA levels in each sample and then to a calibrator consisting of pooled cDNA from multiple samples over the entire time series. Relative quantification of Bmal1 mRNA abundance in all samples was performed using SYBR-Green real-time PCR technology (ABI) as described previously [30,31]. To generate single-strand cDNAs, total RNA (1 mg) from individual samples was reverse transcribed using random hexamers and Superscript III reverse transcriptase Kit (Invitrogen). Real-time PCR analysis was performed on duplicate aliquots using the cDNA equivalent of 1 ng of total RNA for each sample. The PCR cycling conditions were: 1) serial heating at 50uC for 2 min and 95uC for 10 min, 2) amplification over 40 cycles at 95uC for 15 sec and 60uC for 1 min, and 3) dissociation at 95uC for 15 sec, 60uC for 1 min, 95uC for 15 sec and 60uC for 15 sec. To control for differences in sample RNA content, cyclophilin A (Ppia) was amplified with the cDNA equivalent of 1 ng total RNA from the same samples. Consistent with our previous st.