To compare the stability of circular isoform and linear isoform of VRK1, osteosarcoma cells were exposed to 5 μg/ml actinomycin D (Biosharp, Beijing, China) for 8, 16 and 24 hours to block transcription. For RNase R digestion, 2 μg of total RNA was incubated with 8 U RNase R (Sigma-Aldrich, MO, USA) for 30 minutes at 37°C. The levels of transcripts were measured by qRT-PCR analysis [32 (link)].
Rnase r
Manufactured by Merck Group
Sourced in United States
RNase R is a 3'-5' exoribonuclease that efficiently degrades single-stranded RNA. It exhibits robust activity against structured RNA molecules and can be used to remove unwanted RNA species from samples.
Automatically generated - may contain errors
Lab products found in correlation
Actinomycin d, by Merck Group (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Actinomycin d, by R&D Systems (2 mentions)
Rneasy minelute cleanup kit, by Qiagen (2 mentions)
Phenol chloroform, by Merck Group (2 mentions)
Scanner g2505c, by Agilent Technologies (2 mentions)
Actinomycin d, by Biosharp (1 mentions)
Abi 7900, by Thermo Fisher Scientific (1 mentions)
Sybr select master mix kit, by Thermo Fisher Scientific (1 mentions)
Paris kit, by Thermo Fisher Scientific (1 mentions)
Rnase r, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix kit, by Takara Bio (1 mentions)
Primescript reverse transcription kit, by Takara Bio (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Ribo zero rrna removal kit, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
α amanitin, by MedChemExpress (1 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
Quantitect sybr green rt pcr kit, by Qiagen (1 mentions)
41 protocols using rnase r
1
Stability Comparison of Circular and Linear VRK1 Isoforms
2
Circular RNA Profiling and Regulation
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The tissues and cells were isolated in Trizol reagent (Invitrogen), and total RNA samples were obtained. For RNase R treatment, total RNA in cells was incubated with 3 U/μg of RNase R (Sigma-Aldrich, St Louis, MO, USA) for 20 min at 37°C, followed by purification using RNA clean kit (Axygen, Hangzhou, China) according to the instruction. The RNA expression was evaluated by SYBR Select Master Mix kit (Thermo Fisher Scientific, Waltham, MA, USA) with special RT-qPCR primer sets on ABI 7900 (Applied Biosystems, Carlsbad, CA, USA). The sequence of primer sets was circ_0013912: 5ʹ-AGATTGTGCGAACCATGCTC-3ʹ and 5ʹ-CATCATCTGGAATGGGCTCA-3ʹ, miR-7-5p: 5ʹ-CGGCGGTGGAAGACTAGTGATTT-3ʹ and 5ʹ-GTGCAGGGTCCGAGGT-3ʹ, GAPDH, 5ʹ-GACAGTCAGCCGCATCTTCT-3ʹ and 5ʹ-GCGCCCAATACGACCAAATC-3ʹ, and U6: 5ʹ-CTCGCTTCGGCAGCACA-3ʹ and 5ʹ-CTCGCTTCGGCAGCACA-3ʹ. The cycle threshold (Ct) value of each gene was used to examine relative expression of circ_0013912 (GAPDH as internal control) and miR-7-5p (U6 as internal control) using the 2−ΔΔCt method. For subcellular distribution of circ_0013912, Cytoplasmic & Nuclear RNA Purification Kit (BioVision, San Francisco, USA) was utilized to obtain total RNA in cytoplasmic fraction and nuclear fraction, followed with RT-qPCR analysis as above mentioned.
3
Characterizing circZNF652 stability
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For RNase R digestion analyses, total RNA was incubated with 3 U/µg RNase R (#11119915001, Sigma-Aldrich, MO, USA) at 37°C for 30 min. Then, the mRNA levels of circZNF652 and linear ZNF652 were measurement by qRT-PCR. Similarly, total RNA was incubated with 2 mg/mL actinomycin D (R & D Systems, Shanghai, China) for 4, 8, 18, 24 h. And the mRNA levels of circZNF652 and linear ZNF652 were measurement by RT-qPCR.
4
Circular RNA Expression Analysis
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For RNase R treatment, total RNA (2 μg) was incubated at 37 ℃ with or without 3 U/μg of RNase R (Sigma-Aldrich, St. Louis, MO, USA). After treatment with RNase R, qRT-PCR was carried out to evaluate the expression levels of circular-ITCH and linear-ITCH.
For the nuclear and cytoplasmic fraction assay, a PARIS™ Kit (Invitrogen) was employed. Briefly, cells were harvested and lysed in cell fractionation buffer, followed by centrifugation to separate the nuclear and cytoplasmic fractions. The supernatant was transferred to a fresh RNase-free tube. The nuclear pellet was lysed in Cell Disruption Buffer. The cytoplasmic fraction and nuclear lysate were mixed with 2X Lysis/Binding Solution and then incubated with 100% ethanol. The RNAs of nuclear and cytoplasmic fractions were eluted with Elution Solution. U6 and GAPDH were employed as a positive control for nuclear and cytoplasmic fractions, respectively.
For the nuclear and cytoplasmic fraction assay, a PARIS™ Kit (Invitrogen) was employed. Briefly, cells were harvested and lysed in cell fractionation buffer, followed by centrifugation to separate the nuclear and cytoplasmic fractions. The supernatant was transferred to a fresh RNase-free tube. The nuclear pellet was lysed in Cell Disruption Buffer. The cytoplasmic fraction and nuclear lysate were mixed with 2X Lysis/Binding Solution and then incubated with 100% ethanol. The RNAs of nuclear and cytoplasmic fractions were eluted with Elution Solution. U6 and GAPDH were employed as a positive control for nuclear and cytoplasmic fractions, respectively.
5
CircRNA Isolation and Expression Analysis
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TRIzol Total RNA isolation kit (cat. No. KGA1201; Key-Gen Biotech, Nanjing, China) was used for RNA extraction from tissues and cells. Then, cDNA synthesis and quantitative real-time polymerase chain reaction (qPCR) were carried out using a PrimeScript Reverse Transcription Kit (Takara, Shiga, Japan) and SYBR Green Master Mix Kit (Takara), respectively. The 2 -ΔΔCt method was applied for identifying the relative expression of circRNA, miRNA and mRNA using GAPDH or U6 as an endogenous control. Table 2 includes all the primer sequences used in this study.
For RNase R treatment, 2 μg of isolated RNA from GC cells were treated with or without 3 U/mg RNase R (Sigma-Aldrich, St. Louis, USA). After incubation for 60 min at 37°C, the expression of hsa_circ_0017842 and its linear gene FAM188A was measured with qPCR to demonstrate the stability of hsa_circ_0017842.
For RNase R treatment, 2 μg of isolated RNA from GC cells were treated with or without 3 U/mg RNase R (Sigma-Aldrich, St. Louis, USA). After incubation for 60 min at 37°C, the expression of hsa_circ_0017842 and its linear gene FAM188A was measured with qPCR to demonstrate the stability of hsa_circ_0017842.
6
Transcriptomic analysis of circRNA and mRNA
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The circRNA and mRNA sequencing were completed at Novogene Bioinformatics Technology Co. Ltd. (Beijing, China). In this study, total RNA was extracted from the infarcted tissue samples of the sham, MCAO model, and fluoxetine treatment groups using TRIzol (Invitrogen, CA, USA). NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA) was used to evaluate the integrity and concentration of the RNA samples. Ribosomal RNA (rRNA) was removed using Ribo-zero rRNA Removal Kit (Epicenter, Madison, WI, USA). The linear RNA was removed using RNase R (Sigma) for circRNA sequencing. Then, libraries were constructed according to the instruction of NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA). For mRNA sequencing, NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) was used to establish libraries following the kit’s protocol. The circRNA and mRNA libraries were used to perform sequencing on the Illumina Hieq 4000 platform (Illumina, CA, USA).
7
Investigating circTM7SF3 and TM7SF3 mRNA
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Transcription inhibitor Actinomycin D (Sigma; 2 mg/mL; Cat. no. SBR00013) was incubated with THP-1-derived macrophages for 0 h, 4 h, 8 h, 12 h or 24 h, and qRT-PCR was conducted to detect the expression of circTM7SF3 and TM7SF3 messenger RNA (mRNA).
A total of 3 μg RNA sample isolated from THP-1-derived macrophages was incubated with or without 9 units RNase R (Sigma; Cat. no. R6513) for 30 min at room temperature, and the levels of circTM7SF3 and TM7SF3 mRNA were examined by qRT-PCR.
A total of 3 μg RNA sample isolated from THP-1-derived macrophages was incubated with or without 9 units RNase R (Sigma; Cat. no. R6513) for 30 min at room temperature, and the levels of circTM7SF3 and TM7SF3 mRNA were examined by qRT-PCR.
8
Investigating circZBTB44 and HK3 Regulation
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RNase R was used to digest ZBTB44, and α-amanitin was used to inhibit the transcription of HK3. Total RNA was cultured with RNase R (3 U/mg, Sigma-Aldrich) for 20 min at 37 °C. For α-amanitin (Medchemexpress, Shanghai, China) treatment, RCC cells were stimulated with 50 mM α-amanitin for 0, 6, 12, 18, and 24 h. The expression levels of circZBTB44, ZBTB44, HK3 and β-actin were assessed by qRT-PCR analysis.
9
Circular RNA Stability Assay
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To test the stability of circOMA1 expression, actinomycin D (2 mg/ml; Sigma-Aldrich; Merck KGaA) or DMSO was added to inhibit transcription. Total RNA (2 µg) was treated with or without 3U/µg of RNase R (Sigma-Aldrich; Merck KGaA). The resulting RNA was purified with the RNeasy MinElute Cleanup kit (Qiagen, Inc.)
10
RNase R Degradation of RNA
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The RNA of HCT116 and LoVo cells was extracted and incubated with RNase R (Sigma, St Louis, MO, USA). Then, the samples were purified and results were detected with RT-PCR.
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