Northern Blot Analysis of Transcripts

Cell fractionation was performed as described previously (20 (link)). Total RNA was extracted (Nucleospin RNAII; Takara Bio) and poly(A)⁺ RNA was purified (NucleoTrap mRNA Mini kit; Takara Bio) according to the manufacturer's instructions. The RNA samples were loaded on 1.5% gels (NorthernMax-Gly Kit; Life Technologies), transferred to Hybond N+ nylon membranes (GE Healthcare) and probed with internally DIG-labeled sequences following pre-hybridization in ULTRAhyb hybridization buffer (Ambion). DIG-labeled RNA probes were prepared from template DNA amplified by specific primers (Supplementary Table S2) using a DIG Northern Starter Kit (Roche). Visualization of transcripts was performed with a CDP-Star reagent (Roche). Signals were detected by an LAS4000 mini biomolecular imager (GE Healthcare). The densitometric analysis of each band was performed by ImageQuant TL analysis software (GE Healthcare).

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Koyama A., Sugai A., Kato T., Ishihara T., Shiga A., Toyoshima Y., Koyama M., Konno T., Hirokawa S., Yokoseki A., Nishizawa M., Kakita A., Takahashi H, & Onodera O. (2016). Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43. Nucleic Acids Research, 44(12), 5820-5836.

Publication 2016

Nucleospin RNAII NucleoTrap mRNA Mini kit NorthernMax-Gly Kit Hybond N+ nylon membranes ULTRAhyb hybridization buffer DIG Northern Starter Kit CDP-Star reagent LAS4000 mini biomolecular imager ImageQuant TL analysis software

Buffer Cell fractionation Densitometric Gels Hybridization Membranes Mrna Nylon Poly a rna Primers Rna probes

Corresponding Organization :

Other organizations : Niigata University

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Variable analysis

independent variables

Cell fractionation procedure

dependent variables

Abundance of transcripts

control variables

RNA extraction and purification methods
Gel electrophoresis and Northern blotting conditions
DIG-labeling of RNA probes
Hybridization and visualization conditions

positive controls

DIG-labeled RNA probes prepared from template DNA amplified by specific primers

negative controls

Not explicitly mentioned

Annotations

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