For northern blot analysis, small RNA (<200 nucleotides) was extracted from mouse liver or lymph using MirVana. Small RNA was condensed with ethachinmate (Nippon Gene), and 2 μg RNA was separated by electrophoresis on 14% polyacrylamide-urea gels and transferred to Hybond-N + membranes (Amersham Biosciences, Uppsala, Sweden). Blots were hybridized with a fluorescein-labeled probe (Gene Images 3′-Oligolabelling Kit, Amersham Biosciences) for the siRNA antisense sequence or mouse U6 sequence. Signals were visualized using a Gene Images CDP-Star Detection Kit (Amersham Biosciences).
Gene images cdp star detection kit
Manufactured by Cytiva
Sourced in United States
The Gene Images CDP-Star Detection Kit is a laboratory product used for the detection and analysis of DNA or RNA samples. It utilizes a chemiluminescent substrate, CDP-Star, to enable the visualization and quantification of target molecules. The kit provides the necessary reagents and components to perform this detection process.
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3 protocols using gene images cdp star detection kit
1
Northern Blot Analysis of Small RNA
2
Mouse Liver RNA Extraction and Northern Blot
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Total RNA was extracted from mouse liver using Isogen II (Nippon Gene). Total RNA (30 μg) was separated by electrophoresis in an 18% polyacrylamide urea gel and transferred to a Hybond-N+ membrane (Amersham Biosciences, Piscataway, NJ). The blot was hybridized with a probe corresponding to the cRNA sequence, or with the mouse U6 micro-RNA sequence (internal control), which had been labelled with digoxigenin-ddUTP using a DIG Oligonucleotide 3′-End Labelling Kit, 2nd Generation (Roche Diagnostics). The sequence of the DNA probe for detecting cRNA was 5′-TGGTGCGTATGCGTAGCATTGGTATTCA-3′. The signals were visualized using the Gene Images CDP-star Detection Kit (Amersham Biosciences).
3
Northern Blot Analysis of Toc-HDO
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Northern blot analysis of the Toc-HDO with the 36-mer coRNA/coDNA was performed as previously reported with slight modifications.13 (link) Briefly, total RNA was extracted from mouse liver using Isogen II (Nippon Gene, Tokyo, Japan). Thirty micrograms of total RNA was separated by electrophoresis in an 18% polyacrylamide-7 M urea gel and transferred to a Hybond-N+ membrane (Amersham Biosciences, Piscataway, NJ, USA). The blot was hybridized with a probe corresponding to the sequence of the extended portion in coRNA/coDNA, or with a probe to U6 snRNA as a loading control. Both probes were labeled with digoxigenin (DIG)-2′,3′-dideoxyuridine-5′-triphosphate (ddUTP) using a DIG oligonucleotide 3′ end labeling kit, second generation (Roche Diagnostics, Basel, Switzerland). The sequence of the DNA probe for detecting extended 2′OMe was 5′-TGGTGGTGCGTATGCGTAGC-3′. The signals were visualized using a Gene Images CDP-Star detection kit (Amersham Biosciences, Piscataway, NJ, USA), and band intensities were analyzed using Image Lab software version 5.2 of ChemiDoc Touch (Bio-Rad, Hercules, CA, USA).
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