The meiRNA (1–508 nt) was used for in vitro CLIP–seq experiments. The RNA was transcribed and purified in vitro (Lv et al., 2019 (link)). The DNA template used to transcribe the meiRNA was synthesized by TaKaRa Bio Inc. and dissolved in DEPC-treated water to a final concentration of 100 mM. The reaction mixture comprised 10 mM Tris, 10 mM DTT, 10 mM NTPs, 40 mM MgCl2, 0.3 mM T7 template, 0.3 mM DNA templates, and 3 mg/ml T7 polymerase. The reaction was performed at 37°C for 4 h. After transcription, the transcription products were treated with 0.1 total volume (0.1 V) of 0.5 M EDTA, 0.1 V of 5 M NaCl, and 3 V of absolute alcohol and incubated at −40°C overnight. The transcription products were then centrifuged, the supernatant was discarded, and the precipitated RNA was dissolved in 1.5 ml of DEPC-treated water. An equal volume of RNA loading buffer (TaKaRa Bio Inc.) was added, and the mixture was incubated at 90°C for 5 min and cooled on ice for 5 min. The RNA samples were separated on a 12% denaturing polyacrylamide gel and purified using Elutrap (Whatman). The final meiRNA was dialyzed against DEPC-treated water and stored at −80°C.
Rna loading buffer
Manufactured by Takara Bio
Sourced in China
RNA loading buffer is a solution used in molecular biology experiments to prepare RNA samples for analysis. It helps to denature and maintain the structure of RNA molecules, ensuring accurate detection and quantification during electrophoresis or other downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Elutrap, by Cytiva (1 mentions)
Positively charged nylon membrane, by Thermo Fisher Scientific (1 mentions)
Dig labeled lna modified probes, by Qiagen (1 mentions)
Dig luminescent detection kit, by Roche (1 mentions)
Hematoxylin eosin staining kit, by Solarbio (1 mentions)
Pe mouse igg1 κ isotype control, by BioLegend (1 mentions)
Fitc mouse igg1 κ isotype control, by BioLegend (1 mentions)
Apc mouse igg1 κ isotype control, by BioLegend (1 mentions)
Gelred dye, by Biotium (1 mentions)
Depc treated water, by Solarbio (1 mentions)
Fitc mouse anti human cd45, by BioLegend (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Superreal premix plus sybr green, by Tiangen Biotech (1 mentions)
Cck 8, by Beyotime (1 mentions)
Rnasimple total rna kit, by Tiangen Biotech (1 mentions)
Edu staining kit, by Beyotime (1 mentions)
Blocking buffer, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Expresshyb solution, by Takara Bio (1 mentions)
6 protocols using rna loading buffer
1
In Vitro Transcription and Purification of meiRNA
2
Quantification of RNA Expression via Northern Blot
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Northern blots were performed using a DIG luminescent detection kit (Roche) according to the manufacturer’s instructions. Briefly, total RNA (2 μg) was mixed with RNA loading buffer (no. 9168, Takara) and heated at 65°C for 10 min. After cooling on ice for 10 min, the samples were loaded on an agarose (1.5%)-formaldehyde (2.2 M) gel at 60 V for 4 h. The separated RNAs were then transferred to a positively-charged nylon membrane (Thermo Fisher Scientific) and crosslinked with UV light. DIG-labeled LNA-modified probes (Exiqon) complementary to the target genes were hybridized to the membrane at 55°C overnight. The sequences of the probes were as follows: lnc-TSI, 5′-ACATCTCTTAATCAGCGAATCA-3′; ACTB, 5′-CTCATTGTAGAAGGTGTGGTGCCA-3′.
3
Mesenchymal Stem Cell Characterization
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TGF-β1 (PEPRO TECH, China, 100-21-10 μg), PC-MSC (Provided by Shenzhen 150 Biomedical Co.,Ltd.), ESC (Provided by ATCC), FITC Mouse IgG1 κ Isotype control (Biolegend, 7313762), PE Mouse IgG1 κ Isotype control (Biolegend, 9217868), APC Mouse IgG1 κ Isotype control (Biolegend, 9282592), PE Mouse Anti-Human CD73 (Biolegend, 8151901), APC Mouse Anti-Human CD105 (Biolegend, 9277133), FITC Mouse IgG2a κ Isotype control (Biolegend, 8241935), FITC Mouse Anti-Human CD14 (Biolegend, 8299630), FITC Mouse Anti-Human CD45 (Biolegend, 8206704), DMEM/F12 medium (Peiyuan, Shanghai, L310 kJ), Fetal bovine serum (Gemini, 900-108), Trypsin digestive fluid (Beyo, C0203), DPBS (Beyo, C0221G), CCK8 (Beyotime, C0039), EDU staining kit (Beyotime, C0081s), Hematoxylin-eosin staining kit (Solarbio, G1120), DEPC-treated water (Solarbio, R1600), trichloromethane (Sinopharm Chemical Reagent Co. LTD., 10006818), GelRed dye (Biotium, #41003), RNA Loading Buffer (TaKaRa, 9168), Tris-Borate-EDTA Buffer (TBE) 10× Powder, pH8.3 (TaKaRa, T9122), RNAsimple Total RNA Kit (TIANGEN, DP419), All-in-One First-Strand Synthesis Master Mix (Yugong Biolabs, EG15133S), SuperReal PreMix Plus (SYBR Green) (TIANGEN, FP205-02), and Methacrylate anhydride (MAA, 0.1 mL/1 g gelatin), ELISA Kit (Solarbio, SEKH-0052), MateRegen® Gel (2104006, BioRegen Biomedical (Changzhou) Co., Ltd.).
4
rRNA Separation by Gel Electrophoresis
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Bacteria were cultured in LB at 37°C until late logarithmic phase (OD600 = 1). Total RNA was isolated with the RNA prep Pure Cell/Bacteria kit (Tiangen Biotec, Beijing, China). One microgram of the total RNA was mixed with an RNA loading buffer (TaKaRa, Dalian, China) and incubated at 65°C for 10 min, followed by incubation on ice for 10 min. Then the 16S and 23S rRNAs were separated by electrophoresis on a gel made of 0.9% Synergel (BioWorld, USA) and 0.7% agarose in TAE as described previously (67 (link)).
5
Northern Blotting Protocol for RNA Analysis
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Briefly, RNA for Northern blotting (NB) was extracted using TRIzol (Life Technologies) according to the manufacturer’s instructions. RNA (20 μg) was mixed with 2×RNA loading buffer (TAKARA) and EB, denatured at 65°C for 15 minutes and then run on a 1.2% agarose/formaldehyde gel and transferred by capillary action onto a nitrocellulose membrane (Millipore). The nitrocellulose membrane was prehybridized with ExpressHyb solution (Clontech) at 42°C for 2 hours. Probes were produced using a North2South Biotin Random Prime DNA Labeling Kit (Thermo Scientific). Primers used for the production of probes are shown in Table 1 . The membrane was hybridized at 42°C overnight with fresh solution containing the corresponding probe and then washed twice at room temperature for 30 min with wash solution 3 (2×SSC, 0.1% SDS) and once at 42°C for 30 min with wash solution 2 (0.1×SSC, 0.1% SDS). The membrane was then blocked with blocking buffer (catalogue #89880A; Thermo Scientific) at room temperature for 30 min. Finally, the membrane was incubated with IRDye 800-conjugated streptavidin diluted in TBST (1:2500) and imaged on an Odyssey CLx infrared imaging system (Li-COR Biosciences).
6
Northern Blot Analysis of miRNA-143
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Small RNAs (~200 nt) were enriched from Trizol (Thermo Fisher Scientific)-extracted total RNA using miRNeasy Mini kit (QIAGEN), according to the manufacturer’s instruction. Forty micrograms of small RNAs was dissolved in 2 × RNA loading buffer (Takara), heated at 95 °C for 5 min, loaded onto 7 M urea-16% PAGE gel, transferred to Zeta-Probe GT membrane (Bio-Rad), and cross-linked with ultraviolet irradiation (1200 mJ/cm3). The membranes were subjected to hybridization with biotin-labeled DNA oligonucleotide probes for miR-143 and U6 small nucleolar RNA (snRNA) overnight at 42 °C, and the detection of hybridization signal was performed using Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific) and imaged using ECL system (Bio-rad). The biotin-labeled DNA oligonucleotide probe sequences for miR-143 and U6 snRNA (loading control) were 5′-TGA GCT ACA GTG CTT CAT CTC A-3′ and 5′-TGT GCT GCC GAA GCG AGC AC-3′.
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