To detect the RNA duplex formed by GHR sense and antisense RNAs, endogenous ribonuclease protection assay was performed as previously described [16 ,31 ,40 ], with minor modifications, using total RNA from chicken liver and leg muscle tissues, LMH cells and primary myoblasts. To remove genomic DNA and single-strand RNA, the total RNA was treated with DNase I (TaKaRa, Otsu, Japan) at 37°C for 30 min and then digested with RNase A (20 ng/μL; TaKaRa) 37°C for 1 h. Following the RNase A protection assay, we used RT-PCR to detect duplex formation. Five sets of primers were designed for PCR: a first set was targeted to the GHR-AS-EST full-length region of the GHR sense and antisense transcripts, and the other sets were targeted to different overlapping regions of these transcripts (Table S1). PCRs were run in 35 cycles using 25-μL reactions and 2 × Taq PCR Master Mix (Tiangen). Products were checked by 2% agarose gel electrophoresis.
Rnase a
Manufactured by Takara Bio
Sourced in Japan, China, United States, Germany
RNase A is a ribonuclease enzyme that catalyzes the hydrolysis of single-stranded RNA molecules. It is commonly used in molecular biology and biochemistry laboratories for the degradation of unwanted RNA in samples, allowing for the isolation and purification of DNA or other target molecules.
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Lab products found in correlation
Propidium iodide, by Merck Group (26 mentions)
Triton x 100, by Merck Group (21 mentions)
Dnase 1, by Takara Bio (17 mentions)
Accuri c6 flow cytometer, by BD (9 mentions)
Facscalibur flow cytometer, by BD (8 mentions)
Accuri c6, by BD (7 mentions)
Propidium iodide, by Thermo Fisher Scientific (5 mentions)
Facscalibur, by BD (4 mentions)
Facsaria 2 flow cytometer, by BD (3 mentions)
Modfit lt 4, by Verity Software House (3 mentions)
Hinfi, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Keygen Biotech (3 mentions)
Propidium iodide, by Beyotime (3 mentions)
Lsrfortessa, by BD (3 mentions)
Propidium iodide solution, by Merck Group (3 mentions)
Propidium iodide, by BD (3 mentions)
Novaseq 6000 platform, by Illumina (3 mentions)
C10310 edu apollo in vitro imaging kit, by RiboBio (2 mentions)
Dmi8 fluorescent microscope, by Leica (2 mentions)
Fluorescence multi detection microplate reader, by Agilent Technologies (2 mentions)
124 protocols using rnase a
1
Detection of GHR Sense-Antisense RNA Duplex
2
Stability Evaluation of miR-9 Complexes
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miR-9 (20 μM) was mixed with PL-1 at a molar ratio of 20:1 (PL-1: miR-9) and incubated at room temperature for 10 min. For RNase A degradation stability, 1 μL of RNase A (diluted to 1 ng/μL, Takara, Japan) was added and incubated at 37 ºC with time course. For serum stability assay, mouse serum was added at a final concentration 50% (v/v), the samples were incubated at 37 ºC with time course. 3 μL samples were frozen with liquid nitrogen and stocked at -80 ºC. Before loading to agarose gel electrophoresis, the samples were treated with heparin (10 μg) to release intact miR-9. Naked miR-9 was used as a control.
3
Extraction and Preparation of Fungal Spores
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To release spores from asci, the asci were suspended in 5 mL of lyticase (β-glucanase) buffer (50 mM potassium phosphate buffer, pH 7.5, 1.4 M sorbitol), and 20 μL of lyticase (Sigma-Aldrich, St. Louis, MO, USA) stock solution (10,000 U/mL was dissolved in 500 μL of 50% glycerol) was added. After a 3 h incubation at 37 °C, the spores were washed twice with lyticase buffer. Then, the spores were resuspended in water and sonicated with an ultrasonic disruptor (Xinchen Biological Technology, Nanjing, China) to rupture the asci membrane. The sonication conditions were as follows: power—45%; duration—5 min, with cycles of 5 s on and 2 s off. Then, the spores were washed 3 times with 0.5% Tween-20 and 2 times with water. The number of spores was counted with a platelet counter.
Spores treated with high salt were prepared by incubating the spores in 0.6 M NaCl solution for 1 min, and the supernatant was removed. This process was repeated three times and then washed twice with water. To treat the spores with RNase A, 2 × 108 spores suspended in 1 mL of water supplemented with 30 U/mL RNase A (TaKaRa, Tokyo, Japan) were incubated at 37 °C for 1 h. Spores were washed twice with water. To prepare protease-treated spores, 2 × 108 spores suspended in 1 mL of water supplemented with 30 U/mL protease (Sigma-Aldrich) were incubated at 37 °C for 1 h and washed twice with water.
Spores treated with high salt were prepared by incubating the spores in 0.6 M NaCl solution for 1 min, and the supernatant was removed. This process was repeated three times and then washed twice with water. To treat the spores with RNase A, 2 × 108 spores suspended in 1 mL of water supplemented with 30 U/mL RNase A (TaKaRa, Tokyo, Japan) were incubated at 37 °C for 1 h. Spores were washed twice with water. To prepare protease-treated spores, 2 × 108 spores suspended in 1 mL of water supplemented with 30 U/mL protease (Sigma-Aldrich) were incubated at 37 °C for 1 h and washed twice with water.
4
Exosome Inhibition and RNase Treatment
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To block exosomes release, LUAD cells exposed to hypoxic condition were pretreated with GW4869 (an inhibitor of exosome formation and release, Sigma-Aldrich, St. Louis, MO USA) at a concentration of 10 µM for 24 h. For RNase A treatment, exosomes were incubated with RNase A (Takara, Shiga, Japan, final concentration of 10 µg/mL) alone or combined with 0.1 % Triton X-100 for 20 min.
5
Exosomal Delivery of miRNA-24 Mimic
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miR‐24 mimic was loaded into exosomes by electroporation. The loading of exosomes with the miRNA‐24 mimic or a scrambled mimic was performed based on a previously optimized protocol.30 , 31 Briefly, the exosomes were diluted in the P1 primary cell solution (Lonza) at a final concentration of 1 μg/μL. The human miRNA‐24 mimic (RiboBio) or a scrambled miRNA mimic control (500 pmol) was added to 200 μL of exosome sample containing 1 μg/μL exosomal protein. The mixtures were transferred into cold electroporation cuvettes and electroporated using the 4D‐Nucleofector™ system (Lonza). After that the mixture was treated with one unit of RNase A (Takara) for 30 minutes in order to degrade the excess miRNA mimic. RNase A was then deactivated by adding 2 μL RNase inhibitor (Takara), and the exosomes were re‐isolated using the total exosome isolation kit (Life Technology) according to manufacturer's instructions. The final pellet (exosomes) was re‐suspended in PBS, divided into 100 μL aliquots and stored at −80°C.
6
Evaluation of Myoblast Proliferation
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EdU assay: EdU (5-Ethynyl-2′-Deoxyuridine) assay was performed with a C10310 EdU Apollo In Vitro Imaging Kit (Ribobio, Guangzhou, China) after myoblasts were incubated in 50 μmol/L 5-ethynyl-2′-deoxyuridine for 2 h after 22 h betaine treatment. All images were captured with a Leica DMi8 fluorescent microscope (Leica, Wetzlar, Germany) in 200× with 6 random fields in 3 wells per group. The proliferation rate = (EdU positive cells)/(total Hoechst 33,342 stained cells)
Cell cycle analysis: myoblasts were harvested after 24 h betaine treatment and fixed in 70% ethanol overnight at −20 °C. Then, the cells were incubated with 50 μg/mL propidium iodide (Sigma, St. Louis, MO, USA), 10 μg/mL RNase A (Takara, Otsu, Japan), and 0.2% Triton X-100 (Sigma, USA) for 30 min at 4 °C. Cell cycle analysis was performed with a BD AccuriC6 flow cytometer (BD Biosciences, USA) and FlowJo (v7.6) software (Treestar Incorporated, Woodburn, OR, USA).
CCK-8 Assay: CCK-8 (Cell Counting Kit 8) assay was performed in a 96-well plate with 10 μL CCK solutions and incubated for 1 h in the cell incubator after 23 h, 35 h, and 47 h betaine treatment. A Fluorescence/Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA) was used here to measure the absorbance at the wavelength of 450 nm.
Cell cycle analysis: myoblasts were harvested after 24 h betaine treatment and fixed in 70% ethanol overnight at −20 °C. Then, the cells were incubated with 50 μg/mL propidium iodide (Sigma, St. Louis, MO, USA), 10 μg/mL RNase A (Takara, Otsu, Japan), and 0.2% Triton X-100 (Sigma, USA) for 30 min at 4 °C. Cell cycle analysis was performed with a BD AccuriC6 flow cytometer (BD Biosciences, USA) and FlowJo (v7.6) software (Treestar Incorporated, Woodburn, OR, USA).
CCK-8 Assay: CCK-8 (Cell Counting Kit 8) assay was performed in a 96-well plate with 10 μL CCK solutions and incubated for 1 h in the cell incubator after 23 h, 35 h, and 47 h betaine treatment. A Fluorescence/Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA) was used here to measure the absorbance at the wavelength of 450 nm.
7
Propagation and Purification of Phage Vectors
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The T7-wild-type (WT), phage 54 and phage 74 were propagated in E. coli BL21 (18 (link)) containing the pEGFP-C1-HA2-AS eukaryotic expression plasmid to generate T7-WT/pEGFP-C1-HA2-AS, phage 54/pEGFP-C1-HA2-AS and phage 74/pEGFP-C1-HA2-AS particles, respectively. Briefly, 500 mL of freshly cultured E. coli BL21 (OD600 nm=1.0) was infected with phages at a multiplicity of infection (MOI) of 0.001 and agitated until complete lysis of bacteria was observed. DNase I and RNase A (Takara, Dalian Bio, China) were added 30 min before harvesting the phages from the culture medium. The lysate was centrifuged for 15 min at 6000 rpm (Avanti ® J-26 XPI, JLA-162500 Rotor) to separate the bacterial debris from the phage particles. Polyethylene glycol 8000 was added to the supernatant at a final concentration of 10% to allow cross-linking of phage particles. The pellet from the secondary centrifugation was resuspended in 50 mL Tris-buffered saline (TBS) buffer, followed by three extractions with 0.1% Triton-114 to remove endotoxin. The endotoxin residue was assayed using the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (Genscript, China). Purified phage particles were examined by titering, Western-blotting and internalized pEGFP-C1-HA2-AS plasmid DNA was quantified by real-time fluorescent quantitative PCR.
8
Cell Cycle Analysis of Transfected Myoblasts
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After 48 h transfection of gene overexpression vectors, chicken primary myoblasts were collected and fixed in 75% ethanol overnight at -20°C. After ethanol fixation, the cells were stained with 50 μg/mL propidium iodide (Sigma) containing 10 μg/mL RNase A (Takara) and 0.2% (v/v) Triton X-100 (Sigma) for 30 min at 4°C. BD Accuri C6 flow cytometer (BD Biosciences) was subsequently used to analyze the cell cycle with Cell Cycle Analysis Kit (Thermo Fisher Scientific, Waltham, MA, United States), and the data analysis was performed using FlowJo 7.6 software (Verity Software House).
9
GHR-AS Impacts Cell Cycle Analysis
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In order to determine the effects of GHR-AS on the cell cycle distribution, the cells were collected 48 h after siRNA transfection. The cells were washed twice with ice cold PBS and fixed with 70% ice-cold ethanol overnight at −20°C until further processing. After incubation in 50 μg/mL propidium iodide (Sigma Life Science, St. Louis, MO, USA) containing 10 μg/mL RNase A (Takara, Otsu, Japan) and 0.2% (v/v) Triton X-100 (Sigma) for 30 min at 4°C, the cells were analyzed using a FACSAriaII flow cytometer (BD Biosciences, San Jose, CA, USA) and ModFit Lt 4.1 software (Verity Software House, Topsham, ME, USA).
10
Nucleic Acid Determination of Phage
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Following the manufacturer’s instructions, DNAse I, RNAse A, and mung bean nuclease (Takara Biomedical Technology Co., Ltd., Beijing, China) were used to confirm the type of nucleic acid in the phage (Kumar et al. 2021 (link)).
Phage DNA was obtained from the purified phage suspension using a viral RNA/DNA extraction kit (Takara Biomedical Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions.
Phage DNA was obtained from the purified phage suspension using a viral RNA/DNA extraction kit (Takara Biomedical Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions.
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