A dual color Southern blot to visualize two genomes or genic regions simultaneously

Zavala, Anamaria G.; Kulkarni, Amit; Fortunato, Elizabeth A.

doi:10.1016/j.jviromet.2013.12.019

Cited by 7 publications

(4 citation statements)

References 4 publications

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“…Specifically, IR dye-conjugated secondary antibodies have been widely used in western blot analysis (Weldon et al 2008). In addition, several studies have used IR dye-labeled streptavidin or α-DIG antibody in Southern blot and northern blot analyses where probes are biotinylated or DIG-labeled (Zavala et al 2014;MacDiarmid et al 2016). Nonetheless, in these protocols, the IR dye is not covalently attached to the probe and the sensitivity has not been directly compared with 32 P-Northern.…”

Section: Introductionmentioning

confidence: 99%

Near-infrared fluorescent northern blot

Miller

Wei

Fields

et al. 2018

RNA

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Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive thanP-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.

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Section: Introductionmentioning

confidence: 99%

Near-infrared fluorescent northern blot

Miller

Wei

Fields

et al. 2018

RNA

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“…The protocol for dual color Southern blot was optimized based on a previous study by Zavala et al 56 . Following electrophoresis of BamHI digested agar plugs, the gel was washed in: depurination solution (0.25 M HCl) for 20 min, denaturation solution (0.5 M NaOH, 1.5 M NaCl) for 60 min and neutralization solution (0.5 M Tris, 1.5 M NaCl pH 7.5) for 30 min.…”

Section: Methodsmentioning

confidence: 99%

“…The protocol for dual color Southern blot was optimized based on a previous study byZavala et al 56. .…”

mentioning

confidence: 99%

Stable twin rDNA loci form a single nucleolus in brewer’s yeast

Lazar‐Stefanita

Luo

Haase

et al. 2022

Preprint

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The nucleolus is the most prominent membraneless compartment within the nucleus1,2. Nucleoli specialize in ribosomal RNA metabolism and are composed of hundreds of ribosomal DNA (rDNA) repeats that form large chromosomal clusters3–5. High recombination rates between repeats can cause nucleolar dysfunction correlated to genome instability6–8, neurodegeneration9,10 and cell aging11–13. Intriguingly, the evolving architecture of genomes has favored a distinct rDNA organization strategy in which a single rDNA locus per chromosome is situated either near the centromere or the telomere14,15. To understand how organisms may benefit from these genome configurations, we utilized a fused-karyotype strain of Saccharomyces cerevisiae16 to engineer a chromosome with twin chromosome-collinear rDNA loci. We show that these twin-rDNA strains exhibit neither growth defects nor chromosomal abnormalities, and rapidly adapt to maintain rRNA homeostasis. Using imaging and chromosome conformation capture, we found that the twin loci merge into a single subnuclear compartment throughout the cell cycle. Finally, we showed a direct correlation between locus size and position relative to chromosome landmarks in which the centromere-distal locus undergoes size reduction at a higher frequency compared to the centromere-proximal counterpart. This work uncovers an unexpected feature in the structural evolution of the rDNA while introducing a new model to address cellular metabolism as a function of rDNA dosage.

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“…A rotating hybridization oven was used for the pre-hybridization, hybridization, and second post-hybridization wash steps; an orbital shaker was used for the first wash step. After the hybridization step, we washed membranes in 2× SSC/0.1% SDS for 15 min × 2 followed by 0.1× SSC/0.1% SDS for 15 min × 2, blocked with 0.6% gelatin from cold water fish skin 64 (Sigma product number G7041) in 1× PBS for 1 h at room temperature, and placed in mouse monoclonal anti-digoxigenin primary antibody immunoglobulin G (IgG)1kappa (clone 1.71.256; Sigma product number 11333062910) stock concentration 100 μg/mL (use concentration of 1:5,000 = 20 ng/mL PBS/0.05% Tween 20 for 1 h at room temperature in a rotating oven) and then overnight at 4°C. We used secondary antibody goat anti-mouse IRDye 680D (75 ng/mL; LI-COR product number 926-68070) in PBS/0.05% Tween 20 for 1 h at room temperature and imaged blots using a laser scanner (LI-COR Odyssey Cxl) and ImageStudio software (LI-COR).…”

Section: Methodsmentioning

confidence: 99%

Antisense oligonucleotide and adjuvant exercise therapy reverse fatigue in old mice with myotonic dystrophy

Kim

Antoury

et al. 2021

Molecular Therapy - Nucleic Acids

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Patients with myotonic dystrophy type 1 (DM1) identify chronic fatigue as the most debilitating symptom, which manifests in part as prolonged recovery after exercise. Clinical features of DM1 result from pathogenic gain-of-function activity of transcripts containing an expanded microsatellite CUG repeat (CUG exp ). In DM1 mice, therapies targeting the CUG exp transcripts correct the molecular phenotype, reverse myotonia, and improve muscle pathology. However, the effect of targeted molecular therapies on fatigue in DM1 is unknown. Here, we use two mouse models of DM1, age-matched wild-type controls, an exercise-activity assay, electrical impedance myography, and therapeutic antisense oligonucleotides (ASOs) to show that exaggerated exercise-induced fatigue progresses with age, is unrelated to muscle fiber size, and persists despite correction of the molecular phenotype for 3 months. In old DM1 mice, ASO treatment combined with an exercise training regimen consisting of treadmill walking 30 min per day 6 days per week for 3 months reverse all measures of fatigue. Exercise training without ASO therapy improves some measures of fatigue without correction of the molecular pathology. Our results highlight a key limitation of ASO monotherapy for this clinically important feature and support the development of moderate-intensity exercise as an adjuvant for targeted molecular therapies of DM1.

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A dual color Southern blot to visualize two genomes or genic regions simultaneously

Cited by 7 publications

References 4 publications

Near-infrared fluorescent northern blot

Near-infrared fluorescent northern blot

Stable twin rDNA loci form a single nucleolus in brewer’s yeast

Antisense oligonucleotide and adjuvant exercise therapy reverse fatigue in old mice with myotonic dystrophy

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