The Taqman primer probes for EZH2 (Hs01016789_m1), TGFBR1 (Hs00610320_m1), TGFBR2 (Hs00234253), CTGF (Hs01026927_g1), GUSB (Hs00939627_m1), miR-202-5p (002362), pri-miR-663a (Hs03304850_pri), miR-4787-5p (464332_mat), and RNU6B (001093) were purchased from Applied Biosystems (Foster City, CA). Pre-miR-202 lentiviral constructs (HmiR0166-MR03) for overexpressing miR-202-5p and the miRNA scrambled control clone (CmiR0001-MR03) was purchased from GeneCopoeia, Inc (Rockville, MD). The GIPZ lentiviral EZH2 shRNAs (RHS4531-EG2146) were purchased from Dharmacon GE (Lafayette, CO). For knockdown studies, miR-202-5p inhibitors (Assay ID: MH12682) were purchased from Applied Biosystems (Foster City, CA).
Cmir0001 mr03
Manufactured by GeneCopoeia
Sourced in China
CmiR0001-MR03 is a real-time PCR instrument designed for quantitative gene expression analysis. It features multi-channel detection and can accommodate multiple sample types. The core function of this product is to perform sensitive and reliable real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Mmir3445 mr03, by GeneCopoeia (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Rnu6b 001093, by Thermo Fisher Scientific (1 mentions)
Lenti pac hiv expression packaging kit, by GeneCopoeia (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Human cd34 cell nucleofector kit, by Lonza (1 mentions)
Human dermal fibroblast nucleofector kit, by Lonza (1 mentions)
Cell line nucleofector kit r, by Lonza (1 mentions)
Nucleofector 2b device, by Lonza (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Cmir an0001 am03, by GeneCopoeia (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hmir0210 mr03, by GeneCopoeia (1 mentions)
Cmir an0001 sn, by GeneCopoeia (1 mentions)
8 protocols using cmir0001 mr03
1
Profiling miR-202-5p and EZH2 in Cells
2
miR-29a Manipulation in MSCs
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The lentiviral vectors containing miR-29a precursor (RmiR6139-MR03), scramble control (CmiR0001-MR03) were obtained from GeneCopoeia Inc. The lentivirus containing miR-29a inhibitor and its control was obtained from Baoke Bio-Technology Co., Ltd. (Zhengzhou, China).
Lentivirus was generated by using Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia). For the transduction of MSCs with lentivirus, 1×106 MSCs were plated, and 20 µl of virus suspension (at a MOI of 50) was added. The cells were placed for 2 h at 4°C, transferred to a plate, and cultured in 5% CO2 at 37°C for 4 d.
The siRNA lentivirus against REST and its control were obtained from Baoke Bio-Technology Co., Ltd. MSCs were transfected with miR-29a inhibitor for 4 d, then 1×106 MSCs were plated, and 20 µl of virus suspension (at an MOI of 50) was added. The cells were cultured in 5% CO2 at 37°C for 2 d.
Lentivirus was generated by using Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia). For the transduction of MSCs with lentivirus, 1×106 MSCs were plated, and 20 µl of virus suspension (at a MOI of 50) was added. The cells were placed for 2 h at 4°C, transferred to a plate, and cultured in 5% CO2 at 37°C for 4 d.
The siRNA lentivirus against REST and its control were obtained from Baoke Bio-Technology Co., Ltd. MSCs were transfected with miR-29a inhibitor for 4 d, then 1×106 MSCs were plated, and 20 µl of virus suspension (at an MOI of 50) was added. The cells were cultured in 5% CO2 at 37°C for 2 d.
3
Generating Stable miR-133 Expressing Cell Lines
4
Nucleofection of Primary Human Cells
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A Nucleofector™ 2b device (Lonza) was used for nucleofection of human primary cells. IMR90 cells, human myoblasts and human CD34+ hematopoietic cells were nucleofected with the Cell Line Nucleofector™ Kit R (Lonza, catalog # VCA-1001, program X-001), the Human Dermal Fibroblast Nucleofector™ Kit (Lonza, catalog # VPD-1001, program P-022) and the Human CD34+ Cell Nucleofector™ Kit (Lonza, catalog # VPA-1003, program U-008), respectively. Cell numbers for each nucleofection were 2 × 105. We used 4.5 μg target plasmid DNA (expressing sgRNA/Cas9 or sgRNA/Cas9–Klenow) and 0.5 μg GFP-expressing plasmid DNA (CmiR0001-MR03, GeneCopoeia, Inc.) for each nucleofection, where the GFP-expressing plasmid DNA was used as an indicator for nucleofection efficiency. Transfected cells were checked for similar proportions of GFP-positive cells under a fluorescent microscope before further experiments.
5
Silencing FOXF2 and Modulating miR-519a in HCC Cells
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Human HCC cell lines, SMMC-7721 (the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China) and Huh7 cells (the Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China), were cultured in Dulbecco’s modified Eagle medium (DMEM, GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO), 100 U/ml penicillin (GIBCO), and 50 mg/ml streptomycin (GIBCO). HCC cells were maintained in the humidified containing of 5% CO2 incubator at 37 °C.
The targeted sequences for FOXF2 siRNA (Sense; 5′-GAA AAG AUU UCG UCC UCA Att-3′, Anti-sense; 5′-UUG AGG ACG AAA UCU UUU Ctg-3′) or a scrambled oligonucleotide as a negative control were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). MiRNAs vectors, including miR-519a mimics (HmiR0342-MR03), the control vector for miR-519a (CmiR0001-MR03), miR-519a inhibitor (HmiR-AN0588-AM03) and the negative control for the miR-519a inhibitor (CmiR-AN0001-AM03), were obtained from Genecopoeia (Guangzhou, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.
The targeted sequences for FOXF2 siRNA (Sense; 5′-GAA AAG AUU UCG UCC UCA Att-3′, Anti-sense; 5′-UUG AGG ACG AAA UCU UUU Ctg-3′) or a scrambled oligonucleotide as a negative control were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). MiRNAs vectors, including miR-519a mimics (HmiR0342-MR03), the control vector for miR-519a (CmiR0001-MR03), miR-519a inhibitor (HmiR-AN0588-AM03) and the negative control for the miR-519a inhibitor (CmiR-AN0001-AM03), were obtained from Genecopoeia (Guangzhou, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.
6
Regulation of YAP1 by miR-345 in HCC
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miR-345 mimic (HmiR0210-MR03; GeneCopoeia, Inc., Guangzhou, China) and control vector (CmiR0001-MR03; GeneCopoeia) were transfected into MHCC-97H cells while miR-345 inhibitor (HmiR-AN0437-AM01; GeneCopoeia) and negative control vector (CmiR-AN0001-SN; GeneCopoeia) were transfected into Hep3B cells. YAP1 overexpression plasmid and the control plasmid were transfected into Hep3B cells overexpressing miR-345 while YAP1 siRNA and scramble siRNA were transfected into MHCC-97H cells with miR-345 knockdown. Cell transfection of HCC cells were carried out in 6-well plates with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). Cells after transfection were collected for western blot analysis, qRT-PCR, would healing assay, Transwell assay and in vivo experiments.
7
Lentivirus Packaging and Delivery to Mice
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The detailed method of lentivirus packaging and delivery into mice are described in Supplementary Material
8
Lenti-miR-153 Overexpression and Inhibition Plasmids
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Lenti-pre-miR-153, the miR-153-3p overexpression plasmid (RmiR6060-MR03, GeneCopoeia, Guangzhou, China) or negative control (NC) using scrambled RNA oligonucleotides of miR-153 (lenti-mis-pre-miR-153-3p) (CmiR0001-MR03, GeneCopoeia, Guangzhou, China) were generated using the pEZX-MR03 lentiviral transfer vector; while, the miR-153 inhibitor plasmid lenti-AMO-153-3p or NC of AMO-153, using scrambled RNA oligonucleotides (leni-mis-AMO-153-3p), was generated using pEZX-AM03 (RmiR-AN0214-AM03, GeneCopoeia, Guangzhou, China). The titers of these RNA oligonucleotide vectors used for experiments were 1 × 108 transducing U/mL.
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