Five μg of total RNA was used for sRNA Northern blot analysis as described previously [30 (link)]. Briefly, RNA was separated in a 15% denaturing polyacrylamide gel, blotted to Hybond NX membranes (Amersham) and chemically crosslinked. Expression of small RNAs was assessed by hybridisation to a gamma [P32]-labelled (Perkin Elmer, UK) nucleic acid oligonucleotide probe. EtBr staining and U6 northern blotting were used to assess equal loading. ZR small RNA ladder (Zymoresearch) was used to size the bands. DNA oligonucleotides with sequence identical to the target sRNA were used as positive controls and, when possible, the primers were loaded in the gel along with the samples. If membranes were re-probed, they were stripped and exposed for several days to check efficient probe removal before re-probing. Positive control and probe sequences are provided in Additional file 4 .
Hybond nx membrane
Manufactured by Cytiva
Sourced in United States, Italy
Hybond-NX membrane is a nylon-based membrane used in nucleic acid transfer and hybridization applications. It provides a high-binding capacity for both DNA and RNA. The membrane is designed for maximum signal-to-noise ratio and consistent performance.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (16 mentions)
Image one software, by Bio-Rad (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Ultrahyb oligo buffer, by Thermo Fisher Scientific (2 mentions)
Phosphorimager screen, by Fujifilm (2 mentions)
Zr small rna ladder, by Zymo Research (1 mentions)
Typhoon trio variable mode imager, by GE Healthcare (1 mentions)
Storm 840 phosphorimager, by GE Healthcare (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Cl 1000 uv crosslinker, by Analytik Jena (1 mentions)
Pharos fxtm plus molecular imager, by Bio-Rad (1 mentions)
Graph prism 6, by GraphPad (1 mentions)
Hybond n membrane, by Cytiva (1 mentions)
Mirvana kit, by Thermo Fisher Scientific (1 mentions)
Accutarget, by Bioneer (1 mentions)
Ultrahyb buffer, by Thermo Fisher Scientific (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
16 protocols using hybond nx membrane
1
Small RNA Northern Blot Analysis
2
Northern Blotting Analysis of Viral RNA
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Detection of viral RNA by Northern blotting was performed in a manner similar to that previously described, with the following modifications (37 (link)). Briefly, 30 μg of total RNA was resolved by 12% polyacrylamide–7 M urea PAGE in Tris-borate-EDTA (TBE) buffer and transferred onto Hybond NX membranes (Amersham) using an Owl HEP-1 semidry electroblotting system (Thermo Scientific). RNA was chemically cross-linked with EDC cross-linking solution [0.16 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 0.13 M 1-methylimidazole; pH 8.0] for 1 h at 65°C, prior to blocking in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 7% SDS for 1 h at 65°C. Membranes were hybridized with radiolabeled probes against the conserved IAV 5′ vRNA termini (5′-AAAAANNNCCTTGTTTCTACT -3′) and, as a loading control, U6 snRNA (5′-GCCATGCTAATCTTCTCTGTATC -3′) at 30°C for 12 h. Membranes were washed three times with 3× SSC and 0.1% SDS at 30°C for 15 min. Radiolabeled probes were detected by autoradiography using a Typhoon Trio variable-mode imager (GE Healthcare).
3
miRNA Detection in BJAB Cells
4
SARS-CoV-2 Infection in Calu-3 Cells
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Calu-3 cells (5 × 106) were infected with SARS-CoV-2 at MOI 0.05 in T75 flasks for 1 h, after which the inoculum was replaced by fresh media, and cells were incubated for either 24 h or 48 h (mock-treated samples were collected after 48 h). Samples were inactivated for 30 min in 5 mL of TRIzol and RNA was isolated in BSL2+ conditions. Ten to 15 µg of total RNA was separated by 15% urea-PAGE, electrotransferred to Hybond-NX membrane (Amersham), and cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (73 (link)). miRNAs were detected using 32P 5′-radiolabeled DNA probes. Densitometry was performed by using Quantity One.
5
Northern Blot Analysis of Viral RNAs
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Total RNA was extracted using the TRIreagent (Sigma), as previously described [37 (link)]. Ten micrograms of total RNA were separated on a denaturing 12% polyacrylamide gel (8M urea, 1 × MOPS buffer) and transferred to a Hybond NX membrane (Amersham Biosciences, Piscataway, NJ, USA) with semidry electroblotting [38 (link)]. RNA was crosslinked (1200 × 100 μJ/cm2, 254 nm) to the membrane using a CL-1000 UV Crosslinker (UVP, CA, USA). The membrane was hybridized in an ULTRAhyb-Oligo buffer (Thermo Fisher, Waltham, MA, USA) with γ-32P-ATP labelled hybridization probes detecting 5′ and 3′ ends of the HAdV-4 VA RNAI, HAdV-5 VA RNAI, or tRNA-Lysine (Supplementary Table S2 ). After overnight hybridization, the membrane was washed 3× for 10 min at 42 °C in a 3× SSC, 0.5% SDS buffer followed by a single wash with a 1× SSC, 0.5% SDS buffer for 15 min at 42 °C. Radioactive signals were detected using a Pharos FXTM Plus Molecular Imager and analysed using a Quantity One 4.6.9 (Bio-Rad).
6
Northern Blot Analysis of miRNA and snoRNA
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Northern blot analysis was performed as described previously (Guo et al. 2014 (link)). Briefly, total RNAs from BJAB cells transduced with either the empty vector or a HSUR1 mutant were TRIzol isolated and separated by 15% urea-PAGE (10 or 20 µg per lane). The RNA was electrotransferred to Hybond-N+ membrane (Amersham) and UV crosslinked. miRNAs, HSUR1, and U6 were detected using radiolabeled probes (Guo et al. 2014 (link)).
For the anti-Sm IP, the RNA was transferred to Hybond-NX membrane (Amersham), which was then cut at the position of Xylene Cylenol (which comigrates with ∼40 nt-long dsDNA); the bottom was 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) crosslinked for higher miRNA sensitivity (Pall and Hamilton 2008 (link)), while the top was UV crosslinked. Densitometry was performed by using Quantity One. P-values were calculated using Graph Prism 6.
For the anti-Sm IP, the RNA was transferred to Hybond-NX membrane (Amersham), which was then cut at the position of Xylene Cylenol (which comigrates with ∼40 nt-long dsDNA); the bottom was 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) crosslinked for higher miRNA sensitivity (Pall and Hamilton 2008 (link)), while the top was UV crosslinked. Densitometry was performed by using Quantity One. P-values were calculated using Graph Prism 6.
7
MicroRNA Detection via Northern Blotting
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RNA was isolated using either TRIzol (Invitrogen) or the mirVana kit (Ambion), resolved on a 15% urea–polyacrylamide gel, transferred to a Hybond-NX membrane (Amersham) and then crosslinked to the membrane chemically with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (47 (link)). The probes were labeled at the 5′ end with T4 polynucleotide (Takara) and [γ-32P] ATP. 5′ end-radiolabeled oligonucleotide complementary to the indicated miRNA was hybridized to the membrane. The radioactive signals were analyzed using a BAS-2500. Band intensities were quantified by Multi Gauge software. To strip off probes, the blot was incubated with a pre-boiled solution of 0.5% SDS for 15 min. Synthetic miRNA duplexes (AccuTarget) were obtained from Bioneer. The sequences of probes are listed in Supplementary Table S2 .
8
RNA blotting and hybridization analysis
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RNA was loaded onto a denaturing 1% agarose gel and blotted to a Hybond NX membrane (Amersham Biosciences), cross-linked at 80°C for 1 h, and hybridized as previously described in detail (49 (link)). Hybridization probes were generated by 5′-end labeling with γ- (32 (link)) P-ATP DNA oligonucleotides complementary to the 5’ end of either the coding sequence of the IL-6 mRNA, or the coding sequence of IκBα. Signals were detected by exposure of membranes to a PhosphorImager screen (Fuji) followed by signal analysis using the Image One software (BioRad). The sequence of the oligonucleotides used for hybridization are listed in Table S1
9
Immunoprecipitation and Northern Blot Analysis
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Immunoprecipitation of the Flag/HA tagged Ago2 protein was done as described previously [20] (link). For Northern blot analysis, RNA was separated on a denaturing 12% polyacrylamide gel and transferred to a Hybond NX membrane (Amersham Biosciences), chemically crosslinked and hybridized as described [21] (link). Hybridization probes were generated by 5′-end labeling of DNA oligonucleotides with γ-32P-ATP by using T4 Polynucleotide Kinase (NEB). Membranes were hybridized with the probe mixtures containing an equal molar concentration of serotype-specific γ-32P-ATP labeled oligonucleotides (for probe nucleotide sequences see Table S2 ). After overnight hybridization in ULTRAhyb buffer (Ambion), the membrane was washed three times for 10 min at 42°C in 3xSSC, 0.5% SDS followed by a single wash with 1xSSC, 0.5% SDS for 15 min at 42°C. Signals were detected by exposure of membranes to a PhosphorImager screen (Fuji) followed by signal analysis with the Image One software (BioRad).
10
Northern Blotting of Viral siRNAs
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Northern blotting was used to validate the presence of vsiRNAs. Briefly, 2 μg of sRNA from virus-free and virus-infected A. fumigatus isolates were fractionated by electrophoresis through 7 M urea 16% denaturing polyacrylamide gels. Following electrophoresis the RNAs were transferred onto Hybond NX membrane (Amersham) using the semi-dry transfer system (Bio-Rad). Cross-linking was performed using 1-ethyl-3-(dimethylaminopropyl) carbodiimide at 60 °C for 2 h as described previously [45 (link)]. Hybridisation was done at 37 °C overnight using P32 labelled probes which were reverse complement oligonucleotides to the sRNAs of interest. To assess the relative expression levels, quantifications were done three times using ImageQuant software (version 8.1, GE Healthcare Life Sciences) and averages were plotted. The sequences of the probes used and validated sRNAs are listed in Additional file 4 .
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