EMSA was performed using RNA oligos synthesized by Sigma, Life Science and radio-labelled using [γ-32P] ATP and T4 polynucleotide kinase (New England Biolabs). Binding reactions containing labelled RNA probes, together with nuclear extracted obtained from transfected HEK-293 cells, were performed in 1 × binding buffer (10 mM NaCl2, 10 mM Tris pH 8.0, 2 mM MgCl2, 5% glycerol and 1 mM DTT) for 60 min at room temperature before electrophoresis on a 8% polyacrylamide native gel at 100 V for 1.5 h in 0.5 × Tris borate/EDTA buffer at 4 °C. Super-shifts were obtained by adding antibodies specific to U1A, U170K and U1C. A pre-run of the gel (approximately 20 min) was performed before the samples were loaded. Following electrophoresis, the gels were dried on 3MM Whatman paper and exposed on a Cyclone Phosphor screen (Packard). For RNase H protection assays, whole-cell extracts in RSB-100 buffer were incubated with 5 μM antisense DNA oligonucleotide complementary to U1snRNA (5′-CAGGTAAGTAT-3′) and 1.5 U RNase H (Promega) for 30 min at 30 °C. Total RNA was extracted and analysed by northern blotting on a 12% polyacrylamide gel using an internal U1 probe (5′-CAAATTATGCAGTCGAGTTTCCCACATTTG-3′).
Rnase h
Manufactured by Promega
Sourced in United States
RNase H is an enzyme that selectively degrades the RNA strand in RNA-DNA hybrids. It plays a crucial role in various molecular biology techniques involving the manipulation of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Rnasin, by Promega (3 mentions)
Superscript 3, by Thermo Fisher Scientific (3 mentions)
Abi 7900ht, by Thermo Fisher Scientific (2 mentions)
Qs3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Mm00439616 m1, by Thermo Fisher Scientific (2 mentions)
Rnaeasy micro kit, by Qiagen (2 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Tbx21 mm00450960 m1, by Thermo Fisher Scientific (2 mentions)
Primer express 2, by Thermo Fisher Scientific (2 mentions)
Ifnb1 mm00439552 s1, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Gapdh hs02758991 g1, by Thermo Fisher Scientific (1 mentions)
Ifnb1, by Thermo Fisher Scientific (1 mentions)
Rnase h, by New England Biolabs (1 mentions)
Hybond n, by Cytiva (1 mentions)
γ 32p atp and t4 polynucleotide kinase, by New England Biolabs (1 mentions)
Polyattract mrna isolation system, by Promega (1 mentions)
Rna sample preparation kit, by Illumina (1 mentions)
26 protocols using rnase h
1
U1 snRNA-Binding Protein Analysis
2
RNA Oligonucleotide Hybridization and Enzymatic Cleavage
3
Interferon and Inflammation Gene Expression Analysis
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From the globin reduced RNA cDNA was synthesised using High Capacity cDNA Reverse Transcription kit (Applied Biosystems), according to the manufacturer’s instructions followed by RNase H (Promega) treatment for 30 min at 37°C. IFNB1, IL1A, IL6, NFKB1, NFKB2, STAT1, STAT2 and IRF7 gene expression were quantified by real-time PCR (7900HT, Applied Biosystems) using the TaqMan system, and normalised to GAPDH mRNA.
Primer probes used were IFNB1 (Hs01077958_s1); IL1A (Hs00174092_m1); IL6 (Hs00985639_m1); NFKB1 (Hs00765730_m1); NFKB2 (Hs010208901); STAT1 (Hs01013996_m1); STAT2 (Hs01013123_m1); IRF7 (Hs01014809_g1); GAPDH (Hs02758991_g1) (all Applied Biosystems).
Primer probes used were IFNB1 (Hs01077958_s1); IL1A (Hs00174092_m1); IL6 (Hs00985639_m1); NFKB1 (Hs00765730_m1); NFKB2 (Hs010208901); STAT1 (Hs01013996_m1); STAT2 (Hs01013123_m1); IRF7 (Hs01014809_g1); GAPDH (Hs02758991_g1) (all Applied Biosystems).
4
Mapping 18S rRNA Cleavage Sites
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For RNase H cleavage assays, 4 μg total RNAs were denatured at 95°C for 3 min with a reverse probe (5′-TTTACTTCCTCTAGATAGTCAAGTTCGACC-3′; 1 μl at 100 μM) hybridizing in the 3′ end of 18S rRNA (37 (link)). After annealing by cooling down to room temperature for 10 min, the reaction mixture was diluted to 30 μl with a reaction mix containing 1× RNase H reaction buffer, 65 μM DTT, 0.5 U/μl RNasin (Promega), and 50 U RNase H (New England Biolabs), and incubated at 37°C for 30 min. The reaction was then blocked by addition of 0.3 M sodium acetate, pH 5.2, and 0.2 mM EDTA. After phenol extraction and RNA precipitation, the samples were analyzed on a 12% polyacrylamide gel containing urea and TBE buffer. The samples were then electro-transferred to a nylon membrane (Hybond N+; Amersham) and revealed with radiolabeled probes.
3′ RACE analysis was adapted from Kiss and Filipowicz (38 (link)). Primers ITS1-Hs-RACE (5′- CGCGAATTCGATCATTAACGGAGCCCGGAG-3′) and ITS1+2-Hs-RACE (5′-CGCGAATTCACCTGCGGAAGGATCATTAAC-3′), which spans the 18S-ITS1 junction up to nucleotide 13 and to nucleotide 2 in the ITS1 respectively, were used for PCR amplification. The amplified fragments were sub-cloned, automatically sequenced and aligned with Jalview software (39 (link)).
3′ RACE analysis was adapted from Kiss and Filipowicz (38 (link)). Primers ITS1-Hs-RACE (5′- CGCGAATTCGATCATTAACGGAGCCCGGAG-3′) and ITS1+2-Hs-RACE (5′-CGCGAATTCACCTGCGGAAGGATCATTAAC-3′), which spans the 18S-ITS1 junction up to nucleotide 13 and to nucleotide 2 in the ITS1 respectively, were used for PCR amplification. The amplified fragments were sub-cloned, automatically sequenced and aligned with Jalview software (39 (link)).
5
Mouse Gene Expression Analysis
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RNA was extracted using RNAeasy microkit (Qiagen) and reverse transcribed into cDNA using as High Capacity Reverse Transcription kit (Applied Biosystems) according to manufacturer’s instructions, followed by RNaseH (Promega) treatment for 30 min at 37°C. cDNA was analyzed for the expression of the following factors on the 7900HT ABI or QS3 real-time PCR systems (Applied Biosystems) using TaqMan primer probes to mouse Maf, Mm 02581355_s1; Il10, Mm00439616_m1; Tbx21, Mm00450960_m1; Ifng, Mm01168134_m1; Gata3, Mm00484683_m1; Il4, Mm00445260_m1; Rorc, Mm01261019_g1 and Il17a, Mm00439619_m1, Foxp3, Mm 00475162_m1, Il2, Mm 00434256_m1 and Il2ra, Mm 01340213_m1 all from Applied Biosystems. The comparative threshold cycle method with Hrpt1, Mm03024075_m1 as an internal control was used for the normalization of target gene expression.
6
Quantitative RT-PCR Analysis of Rat Neuroendocrine Genes
7
Treating Fractions with D-Loop Structures
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Fractions containing D-loop structures were chosen for this treatment. The procedure described by Chattoraj and Stahl (20) was followed. Briefly, seven microliters of the selected fraction was treated with 1.5 units of RNaseH (Promega Corp., Madison, WI, USA) in 4 μL of a buffer containing 20 mM Hepes KOH, pH 8.0; 10 mM MgCl2; 50 mM KCl; and 1 mM DTT. The mixture was incubated for 20 min at 37 °C. The sample was dialyzed on Millipore filters as above, and an aliquot of the dialysate used for electron microscopy analysis.
8
Quantitative Real-Time PCR Protocol
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Zebrafish embryos and mammalian cells were harvested in Trizol. RNA was isolated using standard Trizol procedures, followed by DNase treatment, purification with Phenol:Chloroform: Isoamyl alcohol (25:24:1, pH 4.5) and resuspension in RNase-free water. 1.5 μg of RNA was reverse transcribed using oligo-dT and Superscript III (Invitrogen). After reverse transcription of RNA, the samples were treated with RNase H (Promega) for 30 min at 37°C. For each qRT-PCR 30 ng of cDNA was mixed with 5 μl of 2X SYBR Green Master Mix (ABS), 0.2 μl of a 10 mM forward and reverse primer each (defrosted once) in a 10 μl reaction. The qRT-PCRs were performed in triplicate (technical) using primers described in S4 Table . Reference genes for relative quantification were mob4 in zebrafish [102 (link)] and TATA-binding protein (TBP) in human cells. Fold-change calculations were performed by the ΔΔCt method. Fold-changes from three biological replicates were used to determine the standard error of means. The p-values were calculated using Welch t-test in the R statistical computing software.
9
RNA-Seq Library Preparation Protocol
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cDNA libraries were constructed using RNA Sample Preparation Kit (Illumia, San Diego, CA) following manufacturer's instructions. Briefly, mRNA was purified from total RNA using polyATtract mRNA isolation systems (Promega, Madison, WI). Fragmentation was carried out using divalent cations under elevated temperature in Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and SuperScript II (Promega, Madison, WI). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H (Promega, Madison, WI). Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. In order to select cDNA fragments of preferentially 200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly). Products were quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system. Finally, the library preparations were sequenced on an Illumina Hiseq 2000 platform which generates 100 bp paired-end reads.
10
Measuring PABPC1 Poly(A) Tail Length
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To assess the Pabpc1 poly(A) tail lengths by northern blot, we mixed total RNA with 0.5 μM DNA oligonucleotides that hybridize to PABPC1 or GAPDH in 15 μL. Where indicated, oligo-dT40 was also included at 0.5 μM. After incubation at 65° for 5 min and chilling on ice, the following components were added to the reaction in 30 μL total volume: 1X RNase H buffer (Promega), 10 mM DTT, 15 ng/μL poly(A) (Sigma), 20 U RNasin (Promega), and 1 U RNase H. The reaction proceeded at 37° for 2 hr and was stopped by addition of 270 μL of G-50 buffer (0.25% SDS, 0.3 M NaOAc, 20 mM Tris pH 6.8, and 2 mM EDTA). RNA was isolated by standard phenol:chloroform:isoamyl alcohol (25:24:1) extraction followed by ethanol precipitation. Northern blots were performed as previously described (Bresson and Conrad, 2013 (link)). RNA probes were generated by incorporation of 32P-UTP into in vitro transcribed RNAs using T7 templates generated by PCR.
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