RNA Extraction and Microarray Analysis of Mouse Brain

Total RNA was extracted from mouse brain using Trizol reagent (Invitrogen), and further purified using RNeasyR Mini Kit (Qiagen) as described previously^{35 (link)}. The quality of RNA was initially assessed by electrophoresis on a 1.5% agarose gel, and further determined by absorption spectrophotometer (Agilent Bioanalyzer 2100 [Agilent, Palo Alto, CA]). cDNAs were synthesized by Low Input Quick Amp Labeling Kit. Cy3-Labeled cRNA was synthesized by in vitro transcription with T7 RNA Polymerase. Following fragmentation, 0.6 μg of cRNA was hybridized for 17 hours at 65 °C on the SurePrint G3 Mouse GE 8 × 60 K Microarray using Gene Expression Hybridization Kit. GeneChips were washed using the Gene Expression Wash Buffers Pack and scanned using Agilent DNA Microarray Scanner (G2565CA). Microarray data was processed using GeneChip Operating Software (Feature Extraction).

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Yamasaki T., Deki-Arima N., Kaneko A., Miyamura N., Iwatsuki M., Matsuoka M., Fujimori-Tonou N., Okamoto-Uchida Y., Hirayama J., Marth J.D., Yamanashi Y., Kawasaki H., Yamanaka K., Penninger J.M., Shibata S, & Nishina H. (2017). Age-dependent motor dysfunction due to neuron-specific disruption of stress-activated protein kinase MKK7. Scientific Reports, 7, 7348.

Publication 2017

Trizol reagent RNeasyR Mini Kit Agilent Bioanalyzer 2100 Agilent DNA Microarray Scanner

Agarose Brain Buffers Cdnas Crna Dna microarray Electrophoresis Gene expression Genechip Hybridization Microarray Mouse T7 rna polymerase Transcription Trizol

Corresponding Organization :

Other organizations : Tokyo Medical and Dental University, Waseda University, Tokyo Women's Medical University, RIKEN Center for Brain Science, Discovery Institute, University of California, Santa Barbara, The University of Tokyo, Kumamoto University, Kanazawa University, Nagoya University, Austrian Academy of Sciences, Institute of Molecular Biotechnology

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Microarray data

control variables

RNA extraction and purification method (Trizol reagent and RNeasy Mini Kit)
RNA quality assessment (agarose gel electrophoresis and absorption spectrophotometry)
CDNA synthesis method (Low Input Quick Amp Labeling Kit)
CRNA labeling method (Cy3-labeling and in vitro transcription with T7 RNA Polymerase)
Hybridization conditions (0.6 μg of cRNA, 17 hours at 65 °C)
Microarray platform (SurePrint G3 Mouse GE 8 × 60 K Microarray)
Microarray washing and scanning (Gene Expression Wash Buffers Pack and Agilent DNA Microarray Scanner)

positive controls

Not specified

negative controls

Not specified

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