For each of three biological replicates, NIH/3T3 cells (passages 4 and 5) were seeded on culture dishes (60 mm; Corning, Corning, NY) and maintained at 37°C and 5% CO2 in Dulbecco's minimum essential medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum (FBS; HyClone, Logan, UT), 3,000 µg/ml glucose and 292 µg/ml L-glutamine. At 48-h intervals, the medium was changed and cultures were expanded 1:3 or 1:4. Prior to experimental analysis, cultures were plated in multiple T-75 flasks. At ~24 and 44 h after plating, the culture medium was changed so as to respectively lower the FBS concentration to 10% and then to 5%. To facilitate cell cycle and circadian oscillation synchronization across cultures, cells were subjected to medium replacement and exposed to serum-free medium (DMEM) containing 15 µM forskolin (Calbiochem, La Jolla, CA) for 2 h. Cultures were rinsed and thereafter maintained in serum-free DMEM.
Extracted molecule
total RNA
Extraction protocol
Immediately after forskolin treatment, cells were harvested from individual flasks (approximate density: 1.3 x 106 cells/cm2) at 6-h intervals for 48 h and total cellular RNA was extracted using RNeasy Midi-Kit protocols (Qiagen, Valencia, CA). RNA extracts from individual samples were treated with on-column DNase-I digestion and concentrated with ethanol precipitations.
Label
Biotin
Label protocol
Prior to analysis, the quality of all NIH/3T3 RNA samples was assessed by electrophoresis on 1% agarose gels containing 0.1 µg/ml ethidium bromide. Labeled cRNAs were produced from purified RNA collected at each time point and hybridized on mouse U74v2 GeneChips. Experimental procedures including double-stranded cDNA synthesis and biotinylated cRNA preparation were conducted according to recommended protocols described in Affymetrix GeneChip Expression Analysis Technical Manual. To assure the quality of labeling and fragmentation efficiency, unfragmented and fragmented cRNA products were analyzed on an Agilent 2100 bioanalyzer prior to hybridization on arrays.
Hybridization protocol
Fragmented biotinylated cRNA (15 µg) was hybridized on Affymetrix GeneChip mouse U74 version 2 arrays at 45°C and 60 rpm in a GeneChip Hybridization Oven 640 (Affymetrix, Santa Clara, CA) for 16 h.
Scan protocol
Following hybridization, arrays were washed and stained using Affymetrix protocols for antibody amplification staining on a GeneChip Fluidics Station 400 in conjunction with Affymetrix Microarray Suite 5.0 software. After a brief wash with a nonstringent buffer, the stained signals on the array were then amplified with a solution containing 3 µg/ml antistreptavidin biotinylated antibody (Vector Laboratories, Burlingame, CA), 1x morpholine ethane sulfonic buffer, 2 mg/ml acetylated BSA, and 0.1 mg/ml normal goat IgG for 10 min followed by a second staining with streptavidin phycoerythrin for 10 min at 25°C. After a final wash with a stringent buffer, the probe array was scanned at the excitation wavelength of 570 nm using an Agilent GeneArray Scanner (Palo Alto, CA). After scanning, each image was first checked for major chip defects or abnormalities during hybridization as a quality control. Arrays were scanned using a global scaling strategy in which the average absolute signal intensity of all arrays was set to an arbitrary target signal intensity of 500 prior to uploading into GeneSpring 7.3 software (Agilent Technologies, Palo Alto, CA).
Description
To screen for output signals that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared relative to SCN2.2 cells in vitro and the rat SCN in vivo. Similar to the circadian profiling of the SCN2.2 and rat SCN transcriptomes, NIH/3T3 fibroblasts exhibited rhythmic fluctuations in the expression of the core clock genes, Per2, Bmal1, and Cry1 and 323 functionally-diverse transcripts (2.6%), many of which regulate cellular communication. Overlap in rhythmically-expressed transcripts among NIH/3T3 fibroblasts, SCN2.2 cells and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared to NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among genes mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase (Nos) in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in co-cultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that the gaseous neurotransmitter nitric oxide may play a key role in SCN pacemaker function. Thus, comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective analysis of circadian signals involved in the coupling of SCN oscillators and regulation of rhythmicity in downstream cells.
Data processing
GeneChip signal intensity data from three biological replicates of NIH/3T3 cells were uploaded into GeneSpring 7.2 for additional processing/filtering [Physiol Genomics. 2007 May 11;29(3):280-9]
