Computational protocol: NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies

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Protocol publication

[…] qRT-PCR was performed as described previously (). Total RNA was prepared from cell culture using TRIzol reagent (Life Technologies). The total RNA (500 ng) or the nascent RNA was reverse transcribed using QuantiTect reverse transcription kit (Qiagen, Venlo, Netherlands). The primers were designed by Primer3 software (www-genome.wi.mit.edu/ftp/distribution/software/) and purchased from Invitrogen. Aliquots of cDNA were subjected to real-time PCR, performed using a Lightcycler 480 SYBR Green I Master (Roche, Basel, Switzerland) according to the manufacturer's protocol. Primers used are shown in Supplemental Table S5.For luciferase reporter assays, the human NEAT1 promoter (hg18 coordinates 64940168-64946905) was amplified from human genomic DNA and inserted into the pGL3 plasmid (Promega, Fitchburg, WI) with KpnI and NheI. pGL3-NEAT1promoter and pGL3-SV40 promoter (Promega) were transfected into HeLa cells with Lipofectamine 2000 (Life Technologies) and 5 μM MG132 or equivalent DMSO added 24 h later. After a 17-h incubation, RNA was harvested and reverse transcribed, and levels of luciferase RNA and GAPDH were measured using qPCR. [...] HeLa cells were nucleofected with GFP ASO or NEAT1 ASO (#12) and incubated for 6, 12, and 24 h. Total RNA was then prepared and labeled with Cy3. Samples were hybridized to a Human Oligo Microarray (G4112F; Agilent, Santa Clara, CA) according to the manufacturer's protocol. Arrays were scanned with a G2565BA Microarray Scanner System (Agilent), and the resulting data were analyzed using the GeneSpring GX software (Agilent). The raw data are available in Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/info/linking.html, accession number GSE45158). […]

Pipeline specifications

Software tools Primer3, GeneSpring GX
Databases GEO
Applications Gene expression microarray analysis, qPCR
Diseases Multiple Endocrine Neoplasia Type 1
Chemicals Adenosine