Mice were housed at 20-22°C, 50-60% humidity, a 12 hours light/dark cycle, and food and water ad libitum. At 6 weeks of age, mice were fasted by removing chow for up to 72 hours before sacrifice (n = 6 per group). The animals were kept in metabolic cages to prevent the consumption of beddings and were kept warm with an infrared lamp starting at 24h. All animals were sacrificed between 9:00 and 10:00 a.m. by cervical dislocation. The liver was removed quickly, weighed, snap-frozen in liquid N2, and stored at -80°C.
Extracted molecule
total RNA
Extraction protocol
Total liver RNA was extracted from frozen tissue with TRIzol (Invitrogen), followed by RNeasy (Qiagen) column purification. The quality of RNA was assessed with the RNA 6000 Nano LabChip® Kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA).
Label
Cy3
Label protocol
20 μg mRNA, pooled from 2 livers (1&2), was reverse transcribed with Cy3-labelled dCTP (Perkin Elmer, Boston, USA), using the Agilent Fluorescent Direct Label Kit.
Mice were housed at 20-22°C, 50-60% humidity, a 12 hours light/dark cycle, and food and water ad libitum. At 6 weeks of age, mice were fasted by removing chow for up to 72 hours before sacrifice (n = 6 per group). The animals were kept in metabolic cages to prevent the consumption of beddings and were kept warm with an infrared lamp starting at 24h. All animals were sacrificed between 9:00 and 10:00 a.m. by cervical dislocation. The liver was removed quickly, weighed, snap-frozen in liquid N2, and stored at -80°C.
Extracted molecule
total RNA
Extraction protocol
Total liver RNA was extracted from frozen tissue with TRIzol (Invitrogen), followed by RNeasy (Qiagen) column purification. The quality of RNA was assessed with the RNA 6000 Nano LabChip® Kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA).
Label
Cy5
Label protocol
Cy5-labeled cDNA produced from RNA pooled from 6 fed animals served as the common reference across all arrays.
Hybridization protocol
Hybridization protocol that acompanies the Agilent's 60-mer Mouse Development (22K) Oligo Microarray G4120A was strictly followed
Scan protocol
Agilent’s dual-laser microarray slide scanner was used. The data were retrieved with Agilent’s Feature Extraction software 6.1.1.
Description
Gene-expression profiling, pathway analysis, and immunohistochemistry were carried out on mouse liver after 0, 12, 24, and 72 hours of fasting to look at the liver's adaptive response to fasting.
Data processing
Value column represents Log(REDsignal/GREENsignal) per feature. Processed signals from Agilent feature extraction file were used.
