To overexpress circAGFG1, the full-length cDNA of circAGFG1 was amplified in 293 T cells and then cloned into over expression vector pLCDH-ciR (Geneseed, Guangzhou, China), which contained a front and back circular frame, while, the mock vector with no circAGFG1 sequence served as a control. To knock down circAGFG1, three siRNAs targeting the back-splice junction site of circAGFG1 and a siRNA-NC were synthesized by Geneseed (Guangzhou, China), after efficiency examination by qRT-PCR, siRNA-3 as the most effective one was used to construct siRNA plasmid vector (Additional file 2 : Figure S1). Then the shRNA against circAGFG1 and negative control shRNA-NC were synthesized and cloned into pLL3.7 vector, named as sh-circ and sh-NC, respectively. All vectors were verified by sequencing. The miRNA mimics and inhibitors were purchased from GenePharma (Shanghai, China). Cell transfections were conducted with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocols. The sequences of siRNAs and shRNAs were listed in Additional file 1 : Table S2.
Mirna mimics and inhibitor
Manufactured by GenePharma
Sourced in China
MiRNA mimics and inhibitors are laboratory reagents used in the study of microRNA (miRNA) functions. miRNAs are small, non-coding RNA molecules that regulate gene expression. MiRNA mimics are synthetic molecules designed to mimic the structure and function of specific endogenous miRNAs, while miRNA inhibitors are designed to block the activity of specific miRNAs. These tools are used by researchers to investigate the biological roles of miRNAs in various cellular processes and disease states.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Lipofectamine imax, by Thermo Fisher Scientific (3 mentions)
Sirnas, by RiboBio (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Socs1 overexpressing lentiviral plasmid, by Hanbio Biotechnology (2 mentions)
P53 antibody, by Beyotime (2 mentions)
Sybr green polymerase chain reaction kit, by Selleck Chemicals (2 mentions)
Triton x, by Vetec (2 mentions)
Dualluciferase reporter vector plasmid, by GenePharma (2 mentions)
Dual luciferase reporter gene uorescence detection kit, by Promega (2 mentions)
3 4 5dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt kit, by Beyotime (2 mentions)
Dulbecco s modi ed eagle medium dmem f12, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Revertaid first strand complementary dna cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Signalway Antibody (2 mentions)
Crystal violet, by Beyotime (2 mentions)
Cell counting kit 8 (cck8), by Beyotime (2 mentions)
22 protocols using mirna mimics and inhibitor
1
Circulating RNA Overexpression and Knockdown
2
siRNA Transfection and miRNA Regulation
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Stable transfection small interfering RNAs (siRNAs) were obtained from RiboBio (Guangzhou, China) and transfected into cells using Lipofectamine iMax (Invitrogen, AL, USA) following the manufacturer's instructions. The human lentivirus miR-331-3p sponge and the SOCS1-overexpressing lentiviral plasmid were purchased from HanBio. miRNA mimics and inhibitors were purchased from GenePharma (Shanghai, China). Transfection efficiency was verified using qRT-PCR.
3
Transfection of RNA regulators
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shRNA, siRNA, and miRNA mimics and inhibitors (GenePharma, Shanghai, China) were transfected into cells using Lipofectamine 2000 (Invitrogen, USA). All primers and oligo sequences are provided in the supplemental material [14 (link)1515 (link).
4
miRNA transfection of MSCs
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miRNA mimics and inhibitors were synthesized by GenePharma (Shanghai, China). RGN-MSCs and RGI-MSCs (2×105 cells/well) were plated in 6-well plate and transfected with miRNA mimics (5 nM), inhibitors (200 nM) or negative controls using Lipofectamine 2000 (Life Technologies).
5
Modulating miR-372-3p Function
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Briefly, chemically synthesized miRNA mimics and inhibitors (GenePharma, Shanghai, China) were used to enhance and inhibit the function of miR-372-3p. Sequences are as follows: miR-372 mimic 5′-AAAGU GCUGCGACAUUUGAGCGUGCUCAAAUGUCGCAGCACUUUUU-3′, and miR-372 inhibitor 5′-ACGCUCAAAUGUCGCAGCACUUU-3′ (both purchased from Shanghai Genepharma Company). After 24 h of inoculation, miR-372-3p was transfected for 24 h using the riboFECT™ CP transfection kit, following manufacturer's protocol (RiboBio, Guangzhou, China).
6
Cervical Cancer Cell Line Culture
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Cervical cancer cell lines, SiHa, HeLa, CaSki, c-41, and c-33A (ATCC; USA), were cultured in RPMI-1640 (Gibco™; Thermo Fisher Scientific, Waltham, MA, USA) containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS), and incubated at 37°C, and 5% CO2. The XLOC_006390 siRNA (5′-UUA AGC UAA CGU UUA CCG CAG TT-3′) and miRNA mimics and inhibitors were purchased from GenePharma (Shanghai, China). Knockdown and overexpression of XLOC_006390 was carried out by transfection with the XLOC_006390 siRNA and recombinant XLOC_006390 pcDNA3.1(+) plasmid, respectively. Both SiHa and Caski cells were plated in 6-well plates for 24 hours and then transfected for 48 hours with the XLOC_006390 siRNA or negative control siRNA using Lipofectamine 2000 (Thermo Fisher Scientific). The transfected cells were used for the following studies.
7
Characterization of Anti-VP3 and Anti-TANK Antibodies
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PGL3-Control vector was kindly provided by Wenhai Feng (China Agricultural University, Beijing, China). Restriction enzyme XbaI was purchased from TaKaRa, Poly(I:C) from Sigma, and HRP-conjugated goat anti-mouse IgG and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG from Ding-Guo (China). Anti-VP3 monoclonal antibody and anti-TANK polyclonal antibodies were developed in our laboratory. All miRNA mimics and inhibitors (chemically-modified and single-stranded RNA oligonucleotide that is reverse complement sequence of mature gga-miR-155) were synthesized by GenePharma Company (Shanghai, China).
8
Lipofectamine-mediated Transfection of RNA Regulators
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Lipofectamine 2000 (Invitrogen, USA) was used to transfect small interfering RNA (siRNA) and miRNA mimics and inhibitors (GenePharma, Shanghai, China) into cells. To construct the overexpression plasmid, the sequence of circRBPMS was cloned into the plenti-ciR-GFP-T2A vector. The corresponding sequences were inserted into a pmirGLO vector (Promega, USA) to synthesize luc reporter plasmids. The miR-330-3p inhibitors and miR-330-3p mimics were purchased from GenePharma (Shanghai, China). Three shRNA molecules were designed to target transcripts of RAI2 and constructed into the pGPU6/GFP/puromycin vector: shRNA-1 (5′-GCTGTGCTCCAGAATTTGTTT-3′), shRNA-2 (5′-GCCACACGGTCATTAAGATGG-3′), and shRNA-3 (5′-GGGAAGAGTCCATGGGAAATG-3′).
9
Modulating H19 and miRNA Expressions
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H19 adenovirus (AD-H19), H19 siRNA (Si-H19), and miRNA mimics and inhibitors were purchased from the Gene Pharma Company (Shanghai, China). We designed three siRNA sequences (coded as Si-H19-595, Si-H19-1484, Si-H19-814) to knock down the expression of H19, and AD-H19 was applied to upregulate the expression of H19. In addition, miR22-5p and miR675-5p mimics were used to upregulate the expression of miRNA, miR22-5p, and miR675-5p inhibitors to downregulate their expressions. When cells reached 70%–80% confluence, they were seeded in 12-well plates and were transfected with AD-H19, Si-H19, and miRNA mimics and inhibitors using Lipofectamine 3000 according to the manufacturer’s instructions. The sequences of Si-H19, AD-H19, miRNA mimics, and inhibitors are listed in Table S4 .
10
CASC11 Overexpression and miRNA Interference
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The overexpression and interfering sequences of CASC11 reported previsouly (10 (link)) were used to construct the CASC11 overexpression plasmid and the intervention lentivirus (GeneChem, Shanghai, China). The miR-646 and miR-381-3p intervention viruses (LV-anti-miR-646 and LV-anti-miR-381-3p) were constructed by GeneChem (Shanghai, China). siRAB11FIP2 and its negative control siNC were purchased from RiboBio (Guangzhou, China); miRNA mimics and inhibitors were provided by GenePharma (Suzhou, China). Lipofectamine 3000 (Invitrogen, USA) was used for cell transfection according to the instructions of the manufacturer.
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