Ultrahyb oligo hybridization buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Lithuania, Sweden

ULTRAhyb-Oligo Hybridization Buffer is a buffer solution designed for use in nucleic acid hybridization procedures. It is formulated to facilitate the efficient and specific binding of oligonucleotides to target sequences.

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Lab products found in correlation

Hybond n membrane, by GE Healthcare (12 mentions) T4 polynucleotide kinase, by New England Biolabs (11 mentions) Trizol reagent, by Thermo Fisher Scientific (7 mentions) Trizol, by Thermo Fisher Scientific (6 mentions) Zeta probe gt membrane, by Bio-Rad (6 mentions) Mirvana mirna isolation kit, by Thermo Fisher Scientific (5 mentions) Hybond n membrane, by Cytiva (5 mentions) Nusieve 3 1 agarose, by Lonza (5 mentions) γ 32p atp, by PerkinElmer (4 mentions) Hybond xl membrane, by Cytiva (4 mentions) Hybond membrane, by Cytiva (4 mentions) Ambion rna extraction kit, by Thermo Fisher Scientific (4 mentions) Ureagel complete to ureagel 8, by National Diagnostics (4 mentions) Riboruler high range and low range rna ladders, by Thermo Fisher Scientific (4 mentions) γ32p atp and t4 polynucleotide kinase, by Thermo Fisher Scientific (3 mentions) Hybond nx nylon membranes, by Cytiva (3 mentions) T4 pnk, by New England Biolabs (3 mentions) Hybond xl nylon membrane, by GE Healthcare (3 mentions) α p32 utp, by PerkinElmer (2 mentions) Hybond n, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

ULTRAhyb buffer (63 citations) ULTRAhyb (60 citations) UltraHyb-Oligo buffer (55 citations) ULTRAhyb hybridization buffer (35 citations) Ultrahyb-Oligo (23 citations) Hybridization buffer (20 citations)

76 protocols using ultrahyb oligo hybridization buffer

Northern Blot Analysis of RNA

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Northern analyses were conducted using established protocols (Wilusz et al., 2012 (link)). Briefly, 15 μg of total RNA from transfected HeLa-JVM cells were separated by 1% formaldehyde agarose gel electrophoresis and capillary transferred overnight to a Hybond N membrane (GE Healthcare), following the manufacturer’s instructions. The membranes were cross-linked with a Stratalinker (Stratagene), using the Optimum Crosslink setting, and incubated overnight with radiolabeled oligonucleotide probes in ULTRAhyb-Oligo Hybridization Buffer (Life Technologies). Blots were washed two times with 2X SSC, 0.5% SDS, and visualized with the GE Healthcare Typhoon Trio imaging system (GE Healthcare). Detailed protocols and probe sequences are in the Supplemental Methods section.
For RNase H treatments, 20 μg of total RNA were mixed with 20 pmol of the TAP-1 antisense oligonucleotide (see Supplemental Methods) and heated for 10 minutes at 65°C. After annealing by slow cooling, the RNA was treated with RNase H (New England BioLabs) for 30 minutes at 37°C and subjected to Northern blot analysis using an 8% polyacrylamide gel. RNAs were electroblotted using the Trans-Blot SD Semi-Dry Transfer cell (Bio-Rad, Hercules, CA) to Hybond N+ membrane (GE Healthcare), UV cross-linked, and incubated with the labeled TAP-2 oligonucleotide probe in ULTRAhyb-Oligo Hybridization Buffer (Life Technologies).

Doucet A.J., Wilusz J.E., Miyoshi T., Liu Y, & Moran J.V. (2015). A 3′ poly(A) tract is required for LINE-1 retrotransposition. Molecular cell, 60(5), 728-741.

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Extraction and Analysis of Small RNAs

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Total RNA was extracted from mid-log phase cells grown at 18°C for 3 days using the hot phenol method in equal volumes of AES buffer (50mM sodium acetate pH 5.3, 10mM DTA, 1% SDS) and acid-phenol. The mixture was incubated at 65°C and vortexed every minute for 5 minutes. Afterward, the slurry was transferred to Maxtract High Density tubes (QIAGEN) and RNA was purified by chloroform extraction. Purified RNA was precipitated using Glycoblue (Thermo Fisher Scientific), sodium acetate pH 5.3 and isopropanol. 5μg of total RNA per sample was loaded per lane in a 1% formaldehyde agarose gel. T7 MAXIscript kit (Ambion) was used to generate α-P³²-UTP (PerkinElmer) labeled RNA probes and hybridizations were carried out using the NorthernMax kit (Ambion).
For probing centromeric small interfering RNAs (siRNAs), siRNAs were purified from exponentially growing cells with mirVana miRNA isolation kit (Thermo Fisher Scientific). 20μg of small RNAs (< 200nt) was resolved on 15% urea-PAGE and electro-transferred to HybondTM-N+ (Thermo Fisher Scientific) membrane in 0.5xTBE for 1 hour at 100V. After UV crosslinking the membrane was probed for siRNAs by hybridization with α-P³²-UTP (PerkinElmer) labeled RNA probes (~50nt) corresponding to dg/dh sequence in UltraHyb-oligo hybridization buffer (Thermo Fisher Scientific).

Vo T.V., Dhakshnamoorthy J., Larkin M., Zofall M., Thillainadesan G., Balachandran V., Holla S., Wheeler D, & Grewal S.I. (2019). CPF Recruitment to Non-canonical Transcription Termination Sites Triggers Heterochromatin Assembly and Gene Silencing. Cell reports, 28(1), 267-281.e5.

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Centromeric siRNA Northern Blot Analysis

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Centromeric small interfering RNAs (siRNAs) northern blot analysis was performed as described previously (Sugiyama et al., 2007 (link)). Small RNAs (< 200 nt) were purified from mid-log phase cells with mirVana miRNA isolation kit (Thermo Fisher Scientific). Twenty μg of small RNAs were resolved on a 15% denaturing acrylamide gel and transferred to HybondTM-N+(Thermo Fisher Scientific) membrane in 0.5x TBE for 1 h at 100V. After UV crosslinking, the membrane was hybridized with α-P³²-UTP (PerkinElmer) labeled RNA probes (~50nt) corresponding to dg sequence in UltraHyb-oligo hybridization buffer (Thermo Fisher Scientific).

Holla S., Dhakshnamoorthy J., Folco H.D., Balachandran V., Xiao H., Sun L.L., Wheeler D., Zofall M, & Grewal S.I. (2019). Positioning heterochromatin at the nuclear periphery suppresses histone turnover to promote epigenetic inheritance. Cell, 180(1), 150-164.e15.

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Detecting Mouse tRNA Transcripts

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TRIzol-purified total RNA was resolved on 10% denaturing urea PAGE gels, and subsequently transferred for 1 hour onto Biodyne B Nylon Membrane in 0.5X TBE at 25 V at room temperature. After transfer, membranes were crosslinked in a UV Stratalinker 2400 cross-linker (Stratagene), prehybridized with 10 ml ULTRAhyb-Oligo hybridization buffer (Thermo Fisher Scientific) for 1 hour at 42°C and then subjected to hybridization with 10 pmol biotin-labeled DNA oligo probes (Integrated DNA Technologies) at 42°C for at least 14 hours. Membranes were then rinsed and washed twice with wash buffer (2X saline sodium citrate with 0.5% (w/v) SDS) at 42°C for 1 hour and analyzed using Chemiluminescent Nucleic Acid Detection Module (89880) (Thermo Fisher Scientific). The probe sequences (table S2) were chosen based on predictions from the GtRNAdb database (tRNAscan-SE analysis of complete genomes (62 (link))) and previously validated mouse tRNA gene families (63 (link)).

Yue T., Zhan X., Zhang D., Jain R., Wang K.W., Choi J.H., Misawa T., Su L., Quan J., Hildebrand S., Xu D., Li X., Turer E., Sun L., Moresco E.M, & Beutler B. (2021). SLFN2 protection of tRNAs from stress-induced cleavage is essential for T cell-mediated immunity. Science (New York, N.Y.), 372(6543), eaba4220.

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Northern Blot Analysis of Small RNA Fraction

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Northern blot analysis was conducted as described elsewhere (46 (link)). In short, the small RNA fraction was isolated from mock- or SFV-infected cells (MOI of 10, at 24 h p.i.) by using the mirVana miRNA isolation kit (Thermo Fisher Scientific). RNA was size fractioned on a 0.5× TBE–7 M urea–15% polyacrylamide gel, transferred to Hybond NX nylon membranes (GE Healthcare), and chemically cross-linked by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma). Small RNAs were probed with a set of DNA oligonucleotides (Table S2) that were 5′ end-labeled with [γ-³²P]ATP (Perking-Elmer) using T4 polynucleotide kinase (New England Biolabs). Hybridization to the oligoprobes was performed overnight at 42°C in Ultrahyb Oligo hybridization buffer (Thermo Fisher Scientific). Membranes were washed twice at 42°C with each of the following three buffers: 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.5% SDS, 2× SSC and 0.2% SDS, and 0.2× SSC and 0.1% SDS. The membrane was exposed to a phosphorimager screen for signal detection.

Varjak M., Maringer K., Watson M., Sreenu V.B., Fredericks A.C., Pondeville E., Donald C.L., Sterk J., Kean J., Vazeille M., Failloux A.B., Kohl A, & Schnettler E. (2017). Aedes aegypti Piwi4 Is a Noncanonical PIWI Protein Involved in Antiviral Responses. mSphere, 2(3), e00144-17.

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Total RNA Isolation and Northern Blotting

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Total RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's instructions. For northern blot, 5–10 μg of total RNA was separated on 1.5% formaldehyde agarose gels in 1 × MOPS buffer and blotted onto Hybond‐N⁺ membranes (GE Healthcare). The probes were labelled with digoxigenin (DIG) according to the manufacturer's protocol (DIG High Prime DNA Labeling and Detection Starter Kit II; Roche). Northern blot procedures were performed as previously described (Jiang et al., 2014 (link)). For small RNA northern blotting, 20 μg of total RNA was separated on a 15% polyacrylamide gel and transferred electrophoretically to Amersham Hybond‐NX membranes (GE Healthcare) using buffer for 1 h (Guo et al., 2012 (link)). Chemical crosslinking was conducted to fix the RNA to the membrane (Pall et al., 2007 (link)). DIG‐labelled probes were synthesized according to the manufacturer's protocol (DIG RNA Labeling Mix; Roche). ULTRAhyb‐Oligo Hybridization Buffer (Thermo Fisher Scientific) was used to optimize the hybridization of probes to siRNA targets.

Yan D., Han K., Lu Y., Peng J., Rao S., Wu G., Liu Y., Chen J., Zheng H, & Yan F. (2023). The nanovirus U2 protein suppresses RNA silencing via three conserved cysteine residues. Molecular Plant Pathology, 25(1), e13394.

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Small RNA Isolation and Detection

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Total RNA was isolated using Trizol (Thermo Fisher Scientific 15596018) as per the manufacturer’s instructions. Small RNAs were separated by 8% denaturing polyacrylamide gel electrophoresis (National Diagnostics EC-833) and electroblotted/UV crosslinked to Hybond N+ membrane (GE Healthcare RPN303B). ULTRAhyb-oligo hybridization Buffer (Thermo Fisher Scientific AM8663) was used as per the manufacturer’s instructions. All oligonucleotide probe sequences are provided in Table S3. Blots were viewed and quantified with the Typhoon 9500 scanner (GE Healthcare) and quantified using ImageQuant (GE Healthcare). Representative blots from ≥3 experiments are shown.

Elrod N.D., Henriques T., Huang K.L., Tatomer D.C., Wilusz J.E., Wagner E.J, & Adelman K. (2019). The Integrator complex attenuates promoter-proximal transcription at protein-coding genes. Molecular cell, 76(5), 738-752.e7.

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Northern Blot Analysis of Viral Small RNAs

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Northern blot analysis was conducted as described previously [29 (link),40 (link)]. In short, total RNA was isolated from the SFV or CHIKV infected Aag2 cells (2×10⁶ cells, MOI 10, 48 hpi) by using Trizol reagent. 15 µg of RNA was size-fractioned on a 0.5× TBE, 7 M Urea, 15% polyacrylamide gel, thereafter transferred to Hybond NX nylon membranes (GE Healthcare, Chicago, IL, USA). RNA was chemically cross-linked to membrane by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma, St. Louis, MO, USA). SFV or CHIKV-specific small RNAs were probed with a set of DNA oligonucleotides (Supplementary Table S1) that were 5′ end-labelled with [³²P] γ-adenosine-triphosphate (Perking Elmer, Waltham, MA, USA) using T4 Polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Hybridization of membranes with oligonucleotide-probes was performed overnight at 42 °C in Ultrahyb Oligo hybridization buffer (Thermo Fisher Scientific). Membranes were washed twice at 42 °C with each of the following three buffers: 2× SSC and 0.5% SDS, 2× SSC and 0.2% SDS, 0.2× SSC and 0.2% SDS. The membrane was exposed to a phosphoimager screen for signal detection and quantification.

Varjak M., Dietrich I., Sreenu V.B., Till B.E., Merits A., Kohl A, & Schnettler E. (2018). Spindle-E Acts Antivirally Against Alphaviruses in Mosquito Cells. Viruses, 10(2), 88.

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RNA Extraction and Oligo Probe Labeling

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Oligo probes were end-labeled using T4 polynucleotide kinase (New England Biolabs, cat#M0201). Total RNA was extracted using TRIzol followed by acid phenol extraction. Hybridization was carried out at 42 °C overnight using ULTRAhyb-Oligo hybridization buffer (ThermoFisher) or Church buffer. Probe information is provided in Supplementary Data 1.

Guh C.Y., Shen H.J., Chen L.W., Chiu P.C., Liao I.H., Lo C.C., Chen Y., Hsieh Y.H., Chang T.C., Yen C.P., Chen Y.Y., Chen T.W., Chen L.Y., Wu C.S., Egly J.M, & Chu H.P. (2022). XPF activates break-induced telomere synthesis. Nature Communications, 13, 5781.

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Quantitative Northern Blot Analysis of snRNAs

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Total RNAs were extracted from fly heads using Trizol reagent (ThermoFisher; #15596026), following the manufacturer’s protocol. RNA quality was checked by gel, and 0.6–3 μg of total RNA was loaded in 15% TBE-urea gel (ThermoFisher; #EC68852BOX). RNAs were then transferred onto nylon membrane (GE HealthCare; #RPN303B) and cross-linked by ultraviolet (UV). The membrane was then prehybridized by UltraHyb Oligo Hybridization Buffer (ThermoFisher; #AM8663) and then hybridized with P³²-labeled probes overnight at 50 °C. To make the probes, DNA oligos were annealed to obtain the template for RNA probes, which were synthesized by in vitro transcription using MAXIscript T7 kit (ThermoFisher; #AM1312), supplemented with P³²-α-UTP. Signals were detected by GE Amersham Typhoon 9410 Imager and analyzed by ImageJ for quantification. Probes used are listed in Supplementary Table 4. The same blots were used to probe 2 different snRNAs: either U1 and U11; U2 and U5; U4 and U7 or U4atac and U12. The uncropped scans of the blots are shown in Supplementary Fig. 8.

Chung C.Y., Berson A., Kennerdell J.R., Sartoris A., Unger T., Porta S., Kim H.J., Smith E.R., Shilatifard A., Van Deerlin V., Lee V.M., Chen-Plotkin A, & Bonini N.M. (2018). Aberrant activation of non-coding RNA targets of transcriptional elongation complexes contributes to TDP-43 toxicity. Nature Communications, 9, 4406.

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