C57BL/6J and DBA/2J mice were sacrificed by cervical dislocation for genomic studies at the times mentioned above. Immediately thereafter, brains were extracted and chilled for 1 min in cold phosphate buffered saline before being microdissected as previously described.22 Excised regions were placed in individual tubes, flash‐frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted from individual nucleus accumbens samples (n = 6 strain/treatment group). The nucleus accumbens was selected for gene expression analyses because of the established role this brain region plays in the negative reinforcing effects of withdrawal.19 RNA quality was assessed by Experion Automated Electrophoresis System (BioRad, Hercules, CA, United States). All samples had RNA integrity (RIN) numbers >8. RNA was then processed and hybridized to Mouse Transcriptome 1.0 arrays (Affymetrix, Santa Clara, CA, United States) according to the manufacturer's protocol.
Mouse transcriptome 1.0 arrays
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mouse Transcriptome 1.0 arrays are a microarray product designed for the comprehensive analysis of the mouse transcriptome. The arrays provide full coverage of the known and predicted mouse genes, allowing for the quantification of gene expression levels across the entire genome.
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Lab products found in correlation
Experion automated electrophoresis system, by Bio-Rad (1 mentions)
Rneasy midi kit, by Qiagen (1 mentions)
Wt plus reagent kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Gc scanner 3000, by Thermo Fisher Scientific (1 mentions)
Picopure rna isolation kit, by Arcturus (1 mentions)
Scanner 3000, by Thermo Fisher Scientific (1 mentions)
Wt ovation pico rna amplification system, by Tecan (1 mentions)
3 protocols using mouse transcriptome 1.0 arrays
1
Nucleus Accumbens Transcriptome Analysis
2
Cardiac Total RNA Extraction and Transcriptome Analysis
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The preparation of total RNA from hearts was performed hearts using the RNeasy Midi Kit (Qiagen, Hilden, Germany). The extraction procedure was conducted in accordance with the manufacturer’s manual. RNA integrity was checked with RNA Nano LabChips on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All samples in this study showed high quality RNA Integrity Numbers (RIN; mean 8.9). RNA was further analyzed by photometric Nanodrop measurement and quantified by fluorometric Qubit RNA assays (Life Technologies).
Synthesis of biotin labeled cDNA was conducted on three replicates of both experimental groups according to the manufacturers´ protocol (WT Plus Reagent Kit; Affymetrix, Inc). Briefly, 200 ng of total RNA were converted to cDNA. After amplification by in vitro transcription and another cDNS synthesis cycle, cDNA was fragmented and biotin labeled by terminal transferase. Finally, cDNA was hybridized to Affymetrix Mouse Transcriptome 1.0 arrays, washed and stained and scanned on the GC Scanner 3000 with G7 update as described in the standard Affymetrix GeneChip protocol (Version 2). Signal summarization using the RMA algorithm as well as statistical analysis were performed using the Affymetrix TAC software. The significance threshold was set to p < 0.05 and 0.01.
Synthesis of biotin labeled cDNA was conducted on three replicates of both experimental groups according to the manufacturers´ protocol (WT Plus Reagent Kit; Affymetrix, Inc). Briefly, 200 ng of total RNA were converted to cDNA. After amplification by in vitro transcription and another cDNS synthesis cycle, cDNA was fragmented and biotin labeled by terminal transferase. Finally, cDNA was hybridized to Affymetrix Mouse Transcriptome 1.0 arrays, washed and stained and scanned on the GC Scanner 3000 with G7 update as described in the standard Affymetrix GeneChip protocol (Version 2). Signal summarization using the RMA algorithm as well as statistical analysis were performed using the Affymetrix TAC software. The significance threshold was set to p < 0.05 and 0.01.
3
Laser Capture Microdissection and Microarray Analysis of CA1 and CA2 Neurons in Amigo2-EGFP Mice
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