Because of the early initiation time of this study, we could only collect 193 miRNAs, and the identities were updated using the latest version of the miRBase database (version 21). The mature miRNA sequences and six control probes (four positive and two negative) were used to produce the customized rice miRNA microarray (Combimatrix Custom Array 4 × 2 K, CA, USA). Each miRNA probe was supplied in triplicate on the microarray, and each control probe contained five copies. For each rice cultivar, 2 μg of the purified small RNAs was employed to prepare the fluorochrome-labeled miRNAs (Cy5 Labeling Kit; Mirus Bio LLC, Madison, WI, USA) according to the manufacturer's instructions. Subsequently, the customized miRNA microarray was hybridized with the Cy5-labeled miRNAs in a 42ºC oven with slow microarray rotation for 4 h. After hybridization, the microarray was washed with a SSPE buffer series with 0.05% Tween-20 according to the manufacturer’s protocol (Combimatrix) and then subjected to image scanning and digitization using a GenePix 4000B scanner and GenePix 4.0 software (Molecular Devices) for further data analysis.
Cy5 labeling kit
Manufactured by Mirus Bio
Sourced in United States
The Cy5 labeling kit is a laboratory tool used for the fluorescent labeling of biomolecules, such as proteins, nucleic acids, or other molecules. The kit provides the necessary reagents and protocols to covalently attach the Cy5 fluorescent dye to the target biomolecule, enabling detection and analysis using appropriate imaging or detection equipment.
Automatically generated - may contain errors
3 protocols using cy5 labeling kit
1
Rice miRNA microarray analysis
2
In Vitro Transcription of Plasmid mRNA
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Each plasmid of interest was digested with the restriction enzyme SpeI to linearize the DNA. After purification, DNA was used as template for in vitro transcription using T7 High Yield RNA Synthesis Kit (New England Biolabs, NEB) in the presence of anti-reverse cap analogue (ARCA, NEB) according to manufacturer's protocol. We routinely obtain 40–50 μg of OVA mRNA from a 20 μl reaction (1:3 GTP:ARCA mole ratio). In vitro transcribed (IVT) mRNA was purified with RNEasykit (Qiagen), quantified by spectrophotometry, and analyzed by agarose gel electrophoresis to confirm the synthesis of full-length mRNA. GFP mRNA was labeled with Cy5 labeling kit (Mirusbio) according to manufacturer's protocol.
3
HBV Ribozyme Activity Assay
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The HBV ribozyme activity assay was carried out as previously detailed.66 (link) Briefly, the HBV RNA substrate was 5′-Cy5 labeled using the Mirus Cy5 labeling kit. The labeled substrate was incubated with a square nanoparticle conjugated with HBV ribozyme for 60 min at 37 °C in buffer containing 20 mM MgCl2, 20 mM NaCl, and 50 mM Tris-HCl (pH 7.5). pRNA conjugated to the HBV ribozyme served as a positive control, and pRNA with a disabled HBV ribozyme was used as a negative control. The cleaved fragments were analyzed on the Typhoon fluorescent imaging system.
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