The largest database of trusted experimental protocols

26 protocols using rnase h

1

U1 snRNA-Binding Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
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′).
+ Open protocol
+ Expand
2

RNA Oligonucleotide Hybridization and Enzymatic Cleavage

Check if the same lab product or an alternative is used in the 5 most similar protocols
For 10µg of RNA, RNA pellet suspended in 10µL of water and 1µM of DNA oligonucleotide was added. The mixture was heated to 65°C for 5 min, then the heat block was allowed to slow cool to 37°C. 1x RNaseH Buffer (20mM Tris-HCl pH 7.5, 100mM KCl, 10mM MgCl2), 10mM DTT, 8U RNaseIn Plus, 0.5U RNaseH (Promega) was added to the RNA mixture. This solution was incubated at 37°C for 1 hour. The reaction was quenched with 180µL of G-50 Buffer (20mM Tris-HCl pH 6.8, 300mM sodium acetate, 2mM EDTA, and 0.25% SDS), phenol:chloroform:iso-amyl alcohol (25:24:1) (PCA) extracted, and ethanol precipitated. Oligonucleotides used are listed in Table S3.
+ Open protocol
+ Expand
3

Interferon and Inflammation Gene Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
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).
+ Open protocol
+ Expand
4

Mapping 18S rRNA Cleavage Sites

Check if the same lab product or an alternative is used in the 5 most similar protocols
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)).
+ Open protocol
+ Expand
5

Mouse Gene Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
6

Quantitative RT-PCR Analysis of Rat Neuroendocrine Genes

Check if the same lab product or an alternative is used in the 5 most similar protocols
Total RNA (250–350 ng) was reverse transcribed using the First-Strand Synthesis SuperMix for RT-qPCR (Invitrogen). cDNA products were treated with RNase H (Promega). PCR was performed in triplicate using iTaq™ Universal SYBR® Green Supermix and the following primers: rat AVP forward 5’-GGGCAGGTAGTTCTCCTCCT-3’, rat AVP reverse 5’-CACCTCTGCCTGCTACTTCC-3’; rat cFos forward 5’-AGCATGGGCTCCCCTGTCA-3’, rat cFos reverse 5’-GAGACCAGAGTGGGCTGCA-3’; rat CRF forward 5’-GAGAAAGGGGAAAGGCAAAG-3’, rat CRF reverse 5’-ATCAGAATCGGCTGAGGTTG-3’; rat GR forward 5’-CACCCATGATCCTGTCAGTG-3’, rat GR reverse 5’-AAAGCCTCCCTCTGCTAACC-3’; rat HPRT forward 5’-GTTCTTTGCTGACCTGCTGGAT-3’, rat HPRT reverse 5’-CCAACACTTCGAGAGGTCCTTT-3’. All primer sets were intron-spanning, with the exception of the GR primer set. RT-negative control reactions were performed to ensure that there was no interfering genomic DNA contamination. All samples were normalized to the hypoxanthine guanine phosphoribosyl transferase 1 (HPRT) housekeeping gene, as it is not altered by EtOH treatment (Przybycien-Szymanska et al., 2010 (link)), and transcript fold changes were calculated using the ΔΔCt method (Livak and Schmittgen, 2001 (link)).
+ Open protocol
+ Expand
7

Treating Fractions with D-Loop Structures

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
8

Quantitative Real-Time PCR Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
9

RNA-Seq Library Preparation Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
10

Measuring PABPC1 Poly(A) Tail Length

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!