transfection control pcmv mir  (OriGene)

Journal: Molecular carcinogenesis

Article Title: miFAST: a novel and rapid microRNA target capture method

doi: 10.1002/mc.22780

Figure Lengend Snippet: a. Experimental procedure for miFAST. Biotinylated miRNA mimics are transfected into cell cultures, and cellular lysates are isolated and separated into total input RNA and pull-down groups. Using IFAST, mRNAs associated with Bi-miR-340 and the RISC complex are extracted and RNA is prepared for analysis. b. Venn diagram of microarray datasets combined to determine high-confidence miR-340 targets in melanoma. Blue circle: mRNAs upregulated obtained from miR-340 inhibition experiments. Red circle: mRNAs downregulated from miR-340 overexpression. Green circle: mRNAs that directly interact with miR-340 identified from Bi-miR-340 experiments. In the center are overlapping targets from each of the datasets in bold, which are considered high-confidence direct miR340 targets.

Article Snippet: Expression vectors and cell culture experiments miRZip TM scrambled hairpin RNA control and miRZip TM 340 microRNA inhibitor constructs (System Biosciences Inc., Mountain View, CA) were used to inhibit hsa-miR-340, and PCMV-MIR340 and PCMV-MIR control microRNA overexpression vectors (Origene, Rockville, MD) were used to overexpress hsa-miR-340.

Techniques: Transfection, Isolation, Microarray, Inhibition, Over Expression

Journal: Molecular carcinogenesis

Article Title: miFAST: a novel and rapid microRNA target capture method

doi: 10.1002/mc.22780

Figure Lengend Snippet: Blue circle: 1,134 overexpressed mRNAs detected from the inhibition of miR-340 function. Red circle: 1,421 downregulated mRNAs detected from the overexpression of miR-340. Green circle: 922 mRNAs directly interacting with miR-340 detected using biotinylated miR-340. Overlapping blue and red circles: 30 targets in common between overexpressed mRNAs and downregulated mRNAs. Overlapping blue and green circles: 13 targets in common between overexpressed mRNAs and mRNAs directly interacting with miR-340. Overlapping red and green circles: 456 targets in common between downregulated mRNAs and mRNAs directly interacting with miR-340. Overlapping blue, red, and green circles: 23 targets in common between overexpressed mRNAs, downregulated mRNAs, and mRNAs directly interacting with miR-340.

Article Snippet: Expression vectors and cell culture experiments miRZip TM scrambled hairpin RNA control and miRZip TM 340 microRNA inhibitor constructs (System Biosciences Inc., Mountain View, CA) were used to inhibit hsa-miR-340, and PCMV-MIR340 and PCMV-MIR control microRNA overexpression vectors (Origene, Rockville, MD) were used to overexpress hsa-miR-340.

Techniques: Inhibition, Over Expression

Journal: Molecular carcinogenesis

Article Title: miFAST: a novel and rapid microRNA target capture method

doi: 10.1002/mc.22780

Figure Lengend Snippet: a. Experimental procedure for miFAST. Biotinylated miRNA mimics are transfected into cell cultures, and cellular lysates are isolated and separated into total input RNA and pull-down groups. Using IFAST, mRNAs associated with Bi-miR-340 and the RISC complex are extracted and RNA is prepared for analysis. b. Venn diagram of microarray datasets combined to determine high-confidence miR-340 targets in melanoma. Blue circle: mRNAs upregulated obtained from miR-340 inhibition experiments. Red circle: mRNAs downregulated from miR-340 overexpression. Green circle: mRNAs that directly interact with miR-340 identified from Bi-miR-340 experiments. In the center are overlapping targets from each of the datasets in bold, which are considered high-confidence direct miR340 targets.

Article Snippet: miRZip TM scrambled hairpin RNA control and miRZip TM 340 microRNA inhibitor constructs (System Biosciences Inc., Mountain View, CA) were used to inhibit hsa-miR-340, and PCMV-MIR340 and PCMV-MIR control microRNA overexpression vectors (Origene, Rockville, MD) were used to overexpress hsa-miR-340.

Techniques: Transfection, Isolation, Microarray, Inhibition, Over Expression

Journal: Molecular carcinogenesis

Article Title: miFAST: a novel and rapid microRNA target capture method

doi: 10.1002/mc.22780

Figure Lengend Snippet: Blue circle: 1,134 overexpressed mRNAs detected from the inhibition of miR-340 function. Red circle: 1,421 downregulated mRNAs detected from the overexpression of miR-340. Green circle: 922 mRNAs directly interacting with miR-340 detected using biotinylated miR-340. Overlapping blue and red circles: 30 targets in common between overexpressed mRNAs and downregulated mRNAs. Overlapping blue and green circles: 13 targets in common between overexpressed mRNAs and mRNAs directly interacting with miR-340. Overlapping red and green circles: 456 targets in common between downregulated mRNAs and mRNAs directly interacting with miR-340. Overlapping blue, red, and green circles: 23 targets in common between overexpressed mRNAs, downregulated mRNAs, and mRNAs directly interacting with miR-340.

Article Snippet: miRZip TM scrambled hairpin RNA control and miRZip TM 340 microRNA inhibitor constructs (System Biosciences Inc., Mountain View, CA) were used to inhibit hsa-miR-340, and PCMV-MIR340 and PCMV-MIR control microRNA overexpression vectors (Origene, Rockville, MD) were used to overexpress hsa-miR-340.

Techniques: Inhibition, Over Expression

Journal: Viruses

Article Title: Application Route and Immune Status of the Host Determine Safety and Oncolytic Activity of Oncolytic Coxsackievirus B3 Variant PD-H

doi: 10.3390/v13101918

Figure Lengend Snippet: Oncolytic activity of PD-H-375 against intra-abdominal growing CT-26 tumors. ( A ) Scheme of miR-375TS region within the genome of PD-H-375TS. Two copies of the miR-375TS, separated by a 6 nucleotide long spacer, were inserted into the 3′-UTR of PD-H 24 bp downstream of the 3D polymerase encoding sequence. The miR-375TS are 100% complementary to the pancreas-specific expressed miR-375. The sequence of miR-375TS is shown in capitals. ( B ) Silencing of PD-H-375TS by miR-375. HEK293T cells were transfected with pCMV-miR-216a or pCMV-miR-375 and infected 48 h later with 0.1 MOI PD-H-375TS. Twenty-four hours later virus titers were determined by plaque assay. Shown are mean values ± SEM from two experiments performed, each in triplicate. Significance: ** p < 0.01. ( C ) Comparison of PD-H-375TS and PD-H replication in CT-26Luc cells. HEK293T cells were seeded in 24-well plates and infected with PD-H or PD-H-375TS at an MOI of 1. Twenty four hours later the viruses were isolated and the virus titers determined by plaque assay. ( D ) Oncolytic activity of CVB3-PD-375TS in tumor model of i.p. growing CT-26Luc tumors. The 3 × 10 5 stable luciferase-expressing CT-26Luc cells were i,p. injected into Balb/C mice. At day 3, 4, and 5 after CT-26 tumor cell injection each 1 × 10 7 pfu PD-H-375TS ( n = 6) or PBS (control) were i.p, injected to mice. Luciferase expression was determined by bioluminescence imaging. The diagrams on the left show the luciferase activity on days 3 and 10 after CT-26Luc inoculation. Right: images of animals after application of luciferase at day 3 and 10 after tumor cell injection. * p < 0.05. ( E ) Kaplan–Meier survival curve. Significance: p = 0.0266.

Article Snippet: HEK293T cells were seeded and transfected the next day, when they reached a confluence of 60% with the control plasmids pCMV-miR-216a (Origene Technologies, Rockville, MD, USA), expressing the miR-216a or pCMV-miR-375 (Origene Technologies) expressing the miR-375, each with PEI Max transfection reagent.

Techniques: Activity Assay, Sequencing, Transfection, Infection, Virus, Plaque Assay, Comparison, Isolation, Luciferase, Expressing, Injection, Control, Imaging