Protoplasts from 1-month-old poplar leaves were isolated following previously described methods [75 (link)]. Briefly, 1-month-old poplar leaves were cut into filaments and then placed into an enzyme solution configured with 2% cellulase and 0.5% pectinase and kept in at 28°C in darkness for 3 hours. The liquid was then subjected to centrifugation and 2 mM MES at pH 5.7, 154 mM NaCl, 125 mM CaCl2,5 mM KCl (W5) was added to dilute the poplar leaf protoplasts. Different combinations of plasmids (pRI101-GFP/ProGbFLS::LUC, ProGbFLS::LUC/35S::GbHY5, ProGbFLS::LUC/35S::GbMYB1, ProGbFLS::LUC/35S::GbHY5/35S::GbMYB1, 35S::nLUC/35S::GbMYB1-nLUC and 35S::GbMYB1-nLUC/35S::GbHY5-nLUC) were co-transformed into the protoplasts and cultured for 16 hours. LUC and REN activity was detected using a GLO-MAX20/20 Fluorescence Detector with a Dual Luciferase Reporter Assay Kit (Promega Corp., Madison, WI, USA).
Glomax 20 20 fluorescence detector
Manufactured by Promega
Sourced in United States, China
The GloMax 20/20 fluorescence detector is a laboratory instrument designed for the detection and quantification of fluorescent signals. It is capable of measuring fluorescence intensity from a variety of sample types, including microplates, cuvettes, and tubes. The GloMax 20/20 utilizes a high-sensitivity photomultiplier tube detector to provide accurate and reliable fluorescence measurements.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase detection kit, by Beyotime (2 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Dual luciferase reporter gene test kit, by Promega (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pgl3 promoter vector, by GenScript (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Pgl3 reporter plasmid, by Promega (1 mentions)
5 protocols using glomax 20 20 fluorescence detector
1
Poplar Leaf Protoplast Isolation and Transformation
2
Validation of miR-98-5p Binding Sites
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As previously reported, the binding sites between miR-98-5p and Xist or miR-98-5p and EDEM1 were predicted by TargetScan and Starbase software, and their interaction was confirmed by the luciferase reporter assay (Data S1 ).6 (link) pGL3 (3577193; Promega, Madison, WI, USA) cloned the target fragments called Xist WT and EDEM1 WT. To create the Mut vectors of lncRNA Xist Mut type and EDEM1 Mut, site-specific mutagenesis was analyzed in the binding site of miR-98-5p and lncRNA Xist. The pRL-TK vector was used as an internal control. HT22 cells were co-transfected with 500 ng pRL-TK vector, 500 ng firefly luciferase expression vector pGL3-Xist, or pGL3-EDEM1 (XIST WT, EDEM1 WT, XIST Mut, or EDEM1 Mut) and 50 nM miR-98-5p mimic or its NC by Lipofectamine 2000. The luciferase activity was assessed in the collected cells after hatching for 48 h according to the protocols of the dual-luciferase reporter gene test kit (E1910) on the GloMax 20/20 fluorescence detector (Promega, Madison, WI, USA), and it was determined as the value of firefly luciferase activity/value of Renilla luciferase activity.
3
Luciferase Assay for miR-598 Binding
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Human THBS2 3′-UTR sequence or the mutant (MUT) sequence of THBS2 3′-UTR, containing the predicted binding sites of miRNA-598, was inserted into the pGL3 promoter vector (Genscript, Nanjing, China). The 293 T cells were seeded in 24-well plates (5 × 105 cells/well) on the day before transfection. Subsequently, the cells were co-transfected with luciferase reporter vectors (0.12 µg) and miR-598 mimic or NC using the Lipofectamine 3000 Reagent (Invitrogen, USA). Luciferase reporter assay was conducted 48 h after transfection according to the manufacturer’s instructions. The Luciferase detection kit (RG005, Beyotime) was used to detect the luciferase activity in a Glomax20/20 fluorescence detector (Promega).
4
Sp5 3'UTR Luciferase Assay
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The artificially synthesized Sp5 3′‐untranslated region (UTR) fragments were introduced into the psiCHECK‐2 vector (Promega Corporation, Madison, WI, USA). The complementary sequence mutation site was designed on the Sp5 wild type (WT), which was inserted into psiCHECK‐2 vector reporter plasmid. The correctly sequenced luciferase reporter plasmid Sp5 3′UTR‐WT (100 ng) and Sp5 3′UTR‐mutant type (MUT) (100 ng) were transfected with miR‐486‐5p mimic and mimic NC (2 nmol/L; Dharmacon Research, Inc, Waltham, MA, USA) into HEK‐293T cell (CRL‐1415; Shanghai Xin Yu Biotech Co., Ltd, Shanghai, China). After 48 hours, cells were collected and lysed. The luciferase activity was detected using a luciferase detection kit (RG005; Beyotime Biotechnology, Shanghai, China) on a Glomax20/20 fluorescence detector (Promega Corporation).
5
Characterizing miR-31-5p Regulatory Interactions
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We PCR amplified the miR-31-5p sequence containing binding sites for wild-type MAGI2-AS3 and TNS1 according to the Starbase and cloned into the pGL3 reporter plasmid (Promega, Beijing, China). Then we constructed target plasmids containing wild-type or mutant MAGI2-AS3 and TNS1 constructs (Thermo Fisher Scientific, Shanghai, China). We cultured T24 cells in a 6-well plate for 24 h, and then transfected them with different combinations of reporter and target plasmids for 48 h. We used the GloMax 20/20 fluorescence detector (Promega, Beijing, China) to detect the fluorescence intensity of the reporter gene plasmid.
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