Total RNA was extracted from the 293T-GFP and 293T-DMEΔ cells 48 h after transfection and transferred onto a Hybond N+ membrane (GE Healthcare). The L1 element (accession no. M19503) was amplified by PCR using the DG2106 and DG2107 primers, and randomly labelled with [α-32P]dCTP (Perkin Elmer) using a Klenow fragment (3′ → 5′ exo−) (New England Biolabs). After hybridization, the membrane was exposed to a phosphorimaging screen (Fujifilm) and the radioactivity was measured with a BAS-5000 Phosphorimager (Fujifilm). Primer sequences are provided in Supplementary Table 1 .
Phosphorimaging screen
Manufactured by Fujifilm
Sourced in Japan
The Phosphorimaging screen is a lab equipment used in the detection and quantification of radioactive signals. It serves as a high-sensitivity sensor that converts radioactive decay into a visible image, enabling the analysis of various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (4 mentions)
γ 32p atp, by Hartmann Analytic (4 mentions)
Dna oligo probes, by Merck Group (4 mentions)
Fla5100 reader, by Fujifilm (3 mentions)
Hybond n membrane, by GE Healthcare (2 mentions)
Multi gauge v2, by Fujifilm (2 mentions)
α 32p dctp, by PerkinElmer (1 mentions)
Bas 5000 phosphorimager, by Fujifilm (1 mentions)
Cl 1000 uv crosslinker, by Analytik Jena (1 mentions)
Trans blot electrophoretic transfer cell, by Bio-Rad (1 mentions)
Phosphoimager, by Bio-Rad (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Image gauge software, by Fujifilm (1 mentions)
Fla 9000 phosphorimager, by Fujifilm (1 mentions)
Typhon fla9500, by GE Healthcare (1 mentions)
T4 pnk, by New England Biolabs (1 mentions)
8 protocols using phosphorimaging screen
1
Detection of Genomic L1 Elements
2
Northern blotting for RNA detection
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Northern blotting was performed as described in Kruszka et al. (66 (link)). Briefly: 30 µg of RNA (per sample) isolated from transfected tobacco leaves was loaded on 8 M denaturing urea polyacrylamide gel (15%) in TBE buffer (0.089 M Tris, 0.089 M boric acid, and 0.002 M EDTA, pH 8.0). RNA was then transferred onto the Amersham Hybond-NX nitrocellulose membrane (GE Healthcare) using a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) and fixed using CL-1000 UV Crosslinker (UVP). Prehybridization and hybridization were performed in hybridization buffer (3.5% SDS, 0.375 M sodium phosphate dibasic, 0.125 M sodium phosphate monobasic) at 42 °C with DNA oligo probes (Sigma) labeled at their 5′ ends with γ32P ATP (Hartmann Analytic). U6 was used as a loading control. After washing, the blots were exposed for up to 3 d to a phosphorimaging screen (Fujifilm) and the results were visualized with the Fujifilm FLA5100 reader (Fujifilm) and quantified using Multi Gauge V2.2 (Fujifilm).
3
Small RNA Detection via Northern Blotting
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A total of 30 μg of RNA was separated by denaturing 8 M urea PAGE (15 %). RNA electrophoresis, blot transfer, and hybridization were performed as previously reported [56 (link)]. DNA oligo probes (Sigma) were 5’ labeled with [γ-32P]ATP (6000 Cimmol–1; Hartmann Analytic GmbH, Germany). Mature miRNA and snoRNA were detected on the same blot. The U6 hybridization signal was taken as a loading control. The Decade™ Markers System (Catalog Number: AM7778) was used as a size marker. The blots were exposed for 5 days to a phosphor imaging screen (Fujifilm) and scanned with a Fujifilm FLA5100 reader (Fujifilm Co., Ltd, Japan). Blots were quantified with Multi Gauge V2.2 software. Probe sequences are listed in Additional file 12 : Table S6.
4
RNA Isolation and Northern Blot Analysis
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A 100mg aliquot of tissue was used for isolation of total RNA enriched in small RNA using a protocol as previously described (Pant et al., 2009 (link); Kruszka et al., 2013 (link)). The quality and quantity of RNA were measured with a NanoDrop ND-1000 spectrophotometer, and RNA integrity was estimated on agarose gels. RNA electrophoresis, blot transfer, and hybridization were performed as previously reported (Kruszka et al., 2013 (link)). DNA oligo probes (Sigma) were 5′ labelled with [γ-32P]ATP (6000 Ci mmol–1; Hartmann Analytic GmbH, Germany). A DNA probe complementary to U6 small nuclear RNA (snRNA) was used, and the U6 hybridization signal was taken as a loading control. The blots were exposed for 1 week to a phosphorimaging screen (Fujifilm) and scanned with a Fujifilm FLA5100 reader (Fujifilm Co., Ltd, Tokyo, Japan). Blots were quantified with Multi Gauge V2.2 software.
5
Northern Blotting for RNA Analysis
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Total RNA was isolated using acidic hot phenol method as previously described74 (link) and Northern blotting was performed according to Karkusiewicz et al.76 (link). Per lane 10 µg of total RNA was resolved on 15% Urea-PAGE and transferred from gel to Hybond N + membrane (GE Healthcare) by electroblotting in 1x TBE, cross-linked by UV radiation (1200 mJ/cm2). The membrane was pre-hybridized in prehyb/hyb buffer (2x SSC, 1X Denhardt’s solution, 0.2%SDS) and hybridized at 37 °C in the same solution with oligonucleotide probes (listed in the Table S2 ) labeled with [γ32P]-ATP by T4 polynucleotide kinase (New England Biolabs). The membrane was washed 3 times for 15 min with wash buffer (2X SSC and 0.2% SDS) at 42 °C and exposed to phosphorimaging screen (Fujifilm). Bands were visualized in phosphoimager (BIORAD) and intensities were quantified using Image Gauge software from Fuji. RNA levels were also estimated by the tRNA-HySeq method, essentially as described previously52 (link), with the modifications as described under supplementary methods (Supplementary File S1).
6
Quantitative microRNA Detection via Northern Blot
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Denaturing 8M urea polyacrylamide gel electrophoresis (PAGE) (15%) was used to separate 90 µg of total RNA. The separated RNA was transferred to Amersham Hybond-NX nitrocellulose (GE Healthcare) and UV-crosslinked. MicroRNAs were visualized with DNA oligo probes (Sigma) 5′ labeled with [γ-32P]ATP (6000 Ci mmol–1; Hartmann Analytic GmbH). A U6 small nuclear RNA (snRNA) hybridization signal was used as a loading control. The blots were exposed to a phosphor imaging screen (Fujifilm), scanned with a Fujifilm FLA5100 reader (Fujifilm Co., Ltd), and quantified with Multi Gauge V2.2 software (Tokyo, Japan). Probes are listed in Supplementary Table 1
7
Dicer-Mediated RNA Substrate Cleavage
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Time-course experiments were performed in 10 μl, containing 5 nM labelled RNA substrate, and 100 nM DicerSOM and DicerΔHEL, respectively, in 30 mM Tris (pH 7.0), 30 mM NaCl, 1 mM DTT, and 2 mM MgCl2 at 37°C. Increasing concentrations (12.5, 25, and 50) of DicerSOM and DicerΔHEL1, respectively, were mixed with 5 nM labelled RNA substrate in 30 mM Tris (pH 7.0), 30 mM NaCl, 1 mM DTT, and 2 mM MgCl2. After 60 min incubation at 37°C, the reactions were stopped with equal volume of 95% formamide, boiled for 5 min, and analyzed on a 20% polyacrylamide gel containing 8 M urea.
After electrophoresis, the gels were exposed for 6-18 hours onto a phosphor imaging screen (Fujifilm). The signal was detected using FLA 9000 phosphorimager (Fujifilm) and analyzed in Multi Gauge v3.2 software.
After electrophoresis, the gels were exposed for 6-18 hours onto a phosphor imaging screen (Fujifilm). The signal was detected using FLA 9000 phosphorimager (Fujifilm) and analyzed in Multi Gauge v3.2 software.
8
Primer Extension Scaffold Assembly
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For assembly of scaffolds used for primer extension experiments, the DNA and RNA oligonucleotides (1 mL, 20 mM each) were mixed and incubated at 95 C for 3 min. The mixture was placed immediately on ice for 1 min. Afterwards, 3 mL of 23 HB buffer were added, and the mixture was incubated at room temperature for at least 10 min. Prior to scaffold assembly, the used RNA was radioactively labelled on the 5 0 -end with [g-32P]ATP (10 mCi/mL, Hartmann Analytic) and T4 PNK (NEB), following purification via PAGE. The annealed scaffold was diluted to 2 mM with transcription reaction buffer (TRB) (20 mM Hepes, pH 7.6, 60 mM (NH 4 ) 2 SO 4 , 10 mM MgSO4, 10% glycerol, 2 mM DTT). 1 mL of diluted scaffold was incubated with 1 mL of Pol III (4 mM, also diluted in TRB) for at least 10 min at RT. Primer extension was initiated via addition of 7 mL TRB, supplemented with NTPs (1 mM each, final concentration). The reaction was incubated at 28 C for 20 min and stopped by adding 3 mL of formamide and boiling for 4 min. The reaction was subjected to PAGE using a denaturing (8M Urea) 17% polyacrylamide TBE gel. Radioactive signal was captured with a phosphor-imaging screen (Fujifilm) and detected with a Typhon FLA9500 (GE Healthcare) instrument.
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