Microarray Analysis Protocol

Microarray analysis was performed as previously described (Pai et al., 2014 (link), Rallis et al., 2013 (link)). In brief, Alexa 555- or 647-labeled cDNA was produced from the RNA, using a Superscript direct cDNA labeling system (Invitrogen) and Alexa 555 and 647 dUTP label mix. The cDNA was then purified using an Invitrogen PureLink PCR Purification system. The cDNA was hybridized to the array using a Gene Expression Hybridization kit (Agilent). The array was an Agilent custom-designed array containing 60-mer oligonucleotides synthesized in situ on the array and contained 4 × 44,000 probes. Following hybridization for at least 17 hr, the array was washed using a Gene Expression Wash Buffer kit (Agilent) and scanned in an Agilent Array Scanner. The microarray signal was extracted using GenePix.

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Pai C.C., Kishkevich A., Deegan R.S., Keszthelyi A., Folkes L., Kearsey S.E., De León N., Soriano I., de Bruin R.A., Carr A.M, & Humphrey T.C. (2017). Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription. Cell Reports, 20(11), 2693-2705.

Publication 2017

Superscript direct cDNA labeling system PureLink PCR Purification system Gene Expression Hybridization kit custom-designed array Gene Expression Wash Buffer kit

Buffer Cdna Dutp Gene expression Hybridization Microarray Oligonucleotides

Corresponding Organization : University of Oxford

Other organizations : MRC Laboratory for Molecular Cell Biology, University College London, University of Sussex

Top 5 similar protocols

Variable analysis

independent variables

Alexa 555- or 647-labeled cDNA

dependent variables

Microarray signal

control variables

Superscript direct cDNA labeling system (Invitrogen)
Alexa 555 and 647 dUTP label mix
Invitrogen PureLink PCR Purification system
Gene Expression Hybridization kit (Agilent)
Agilent custom-designed array containing 60-mer oligonucleotides synthesized in situ
Gene Expression Wash Buffer kit (Agilent)
Agilent Array Scanner
GenePix

controls

Positive controls not explicitly mentioned.
Negative controls not explicitly mentioned.

Annotations

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