Northern blots using oligonucleotide probes and NorthernMax reagents (Thermo Fisher Scientific) were performed as previously described in detail [41 (link)]. Probes used for Drosophila cell experiments: Laccase2 Exon 2 XmaI (5′-GCTGAGCTCCCCGGG-3′), Drosophila β-actin (5′-AGCACAGTGTTGGCGTACAG-3′), and HygroR (5′-GACATATCCACGCCCTCCTA-3′). Probes used for human cell experiments: Laccase2 Exon 2 (5′-GCTAGGATTGAGGATGGAGCTCC-3′), cGFP (5′-TCCATGCCGTGGGTGATGCC-3′), and human β-actin (5′-AGCACTGTGTTGGCGTACAG-3′). All blots were viewed with a Typhoon 9500 scanner (GE Healthcare) and quantified using ImageQuant.
Northernmax reagents
Manufactured by Thermo Fisher Scientific
NorthernMax reagents are a set of solutions used for Northern blot analysis, which is a technique used to detect and quantify specific RNA molecules in a sample. The reagents include solutions for sample preparation, electrophoresis, transfer, and detection, enabling researchers to perform this analytical method effectively.
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6 protocols using northernmax reagents
1
Northern Blotting with Oligonucleotide Probes
2
Northern Blot Analysis of RNA
3
Quantitative Northern Blot Analysis
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Northern blots using NorthernMax reagents (ThermoFisher Scientific) and oligonucleotide probes were performed as described previously (Tatomer et al. 2017 (link)). All probe sequences are in Supplemental Table S5 . Blots were viewed with a Typhoon 9500 scanner (GE Healthcare) and quantified using ImageJ. Representative blots are shown.
4
Northern Blot Analysis of Guide RNAs
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Northern blots using 1.2% denaturing formaldehyde agarose gels, NorthernMax reagents (Thermo Fisher Scientific), and oligonucleotide probes were performed as previously described (44 (link)). Oligonucleotide probe sequences are provided in Supplementary Table S3 . Blots were hybridized with individual oligonucleotide probes, washed, and viewed with the Typhoon 9500 scanner (GE Healthcare) followed by quantification using ImageQuant (GE Healthcare). They were then subsequently re-probed with additional oligonucleotide probes so that all blots shown in a figure panel are derived from the same experiment. To confirm expression of the guide RNAs, 8% polyacrylamide gels rather than denaturing formaldehyde agarose gels were used.
5
Northern Blot Assay for HeLa Cells
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HeLa cells were seeded in 12-well plates with 1.5 mL of medium. After 24 h, cells (∼50% confluency) were transfected with 500 ng of plasmid (250 ng of nLuc reporter, 250 ng of pGL4.13, which served as a transfection control) using Viafect (Promega) at 2:1 (reagent:DNA). Twenty-four hours after transfection, total RNA was harvested with TRIzol (Thermo Fisher Scientific). Ten micrograms of total RNA was separated on a 1.2% denaturing formaldehyde agarose gel using NorthernMax reagents (Thermo Fisher Scientific). Northern blots were then performed as described previously (Tatomer et al. 2017 (link)).
6
Northern Blot Analysis of RNA
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Northern blots using NorthernMax reagents (ThermoFisher Scientific) and oligonucleotide probes were performed as described previously (44 (link)). All probe sequences are provided in Supplementary Table S1 . Blots were viewed with the Typhoon 9500 scanner (GE Healthcare). Representative blots are shown.
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