The LNA-anti miR-29a and scramble control on a phosphothiorate background were purchased from Exiqon. Lethally irradiated recipient F1 mice transplanted with BM (5×106) + B6 WT spleen cells (20×106) were treated with LNA-anti miR-29a (n=10) or scramble LNA oligos (n= 10) starting at day 7, at a dose of 10 mg/kg i.p. twice weekly up to day 50 after infusion of donor B6 splenocytes. Clinical GVHD scores and survival was followed post-transplant as described earlier. One cohort of mice were euthanized day 10 post-transplant to evaluate serum miR-29a expression and another to evaluate DC and T cell activation, proliferation, effector status.
Scramble control
Manufactured by Qiagen
The Scramble control is a laboratory reagent used as a negative control in gene expression analysis experiments. It is designed to have no known target in the genome, and is used to assess non-specific signal or background noise in the experimental system.
Automatically generated - may contain errors
Lab products found in correlation
Apotome, by Zeiss (2 mentions)
Hepatocyte culture medium, by Lonza (2 mentions)
Mir 125b 5p mimic, by Qiagen (2 mentions)
Mir 125b 5p inhibitor, by Qiagen (2 mentions)
Liberase, by Roche (2 mentions)
Nbt bcip solution, by Roche (1 mentions)
Axioimager brightfield microscope, by Zeiss (1 mentions)
Anti dig ap antibody, by Roche (1 mentions)
Rpmi 1640, by Qiagen (1 mentions)
Lipofectamine 2000, by Qiagen (1 mentions)
Rpmi 1640, by Biosera (1 mentions)
Recombinant epidermal growth factor, by Lonza (1 mentions)
5 protocols using scramble control
1
Modulating miR-29a in GVHD Transplant
2
In Situ Hybridization of miR-205 in Thymus
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In situ hybridization was performed on frozen thymic sections. At harvest, thymi were fixed for 2 hours in 4% paraformaldehyde and equilibrated for 7 hours in 30% sucrose. We used double DIG labeled LNA probes against either miR-205 or a scramble control (Exiqon). We followed the manufacturer’s instructions and hybridized the probes overnight at 57°C with the following modifications: Post-hybridization stringency washes: 2x SSC for 60’ at 57°C, 1x SSC for 10’ at RT, 0.5x SSC for 10’ at RT, 0.1x SSC for 45’ at 57°C. Tissues were then blocked with 1% goat serum in 0.1% PBS-Tween-20 (PBST) for 2 hours before overnight incubation with a 1:5000 dilution of anti-DIG-AP antibody (Roche) at 4°C. Following antibody incubation and overnight washes in PBST, alkaline phosphatase activity was detected using an NBT/BCIP solution (Roche). Slides were visualized using either a Zeiss Apotome or a Zeiss AxioImager brightfield microscope.
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In situ hybridization was performed on frozen thymic sections. At harvest, thymi were fixed for 2 hours in 4% paraformaldehyde and equilibrated for 7 hours in 30% sucrose. We used double DIG labeled LNA probes against either miR-205 or a scramble control (Exiqon). We followed the manufacturer’s instructions and hybridized the probes overnight at 57°C with the following modifications: Post-hybridization stringency washes: 2x SSC for 60’ at 57°C, 1x SSC for 10’ at RT, 0.5x SSC for 10’ at RT, 0.1x SSC for 45’ at 57°C. Tissues were then blocked with 1% goat serum in 0.1% PBS-Tween-20 (PBST) for 2 hours before overnight incubation with a 1:5000 dilution of anti-DIG-AP antibody (Roche) at 4°C. Following antibody incubation and overnight washes in PBST, alkaline phosphatase activity was detected using an NBT/BCIP solution (Roche). Slides were visualized using either a Zeiss Apotome or a Zeiss AxioImager brightfield microscope.
3
miR-185 Knockdown in THP-1 Macrophages
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THP-1 cells were cultured in RPMI-1640 (Biosera) supplemented with 10% fetal calf serum (FCS) (Biosera) and 1% penicillin-streptomycin in a humidified incubator at 37°C containing 5% CO2. 5 × 105 THP-1 cells were treated with PMA at 50ng/ml final concentration in 2% FCS supplemented RPMI-1640 for 16 hours, followed by addition of Lipofectamine 2000 (complexes with 25 or 12.5 pmol of miR-185 specific antagomir (Exiqon) or scramble control (Exiqon) in serum free RPMI-1640 for 4 hours. Subsequently cells were supplemented with RPMI-1640 containing 2% FCS for 24 hours prior to cell lysis for total RNA extraction or total protein analysis.
4
Isolation and Transfection of Primary Hepatocytes
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Freshly isolated primary human hepatocytes were purchased from Cytonet GmBH. Primary hepatocytes from mouse liver were isolated as described26 (link). Briefly, mice were anaesthetized and perfused with Liberase (Roche). After perfusion, livers were disintegrated mechanically before collecting hepatocytes by low-speed centrifugation. Non-parenchymal cells were removed by discarding the supernatant. For all in vitro transfection experiments, we used Percoll density gradient-purified mouse hepatocytes to achieve high transfection efficiency. Ten thousand primary hepatocytes per well of a collagen-coated 12-well plate (BD) were seeded. Twelve hours after seeding, hepatocytes were transfected with 25 nM miR-125b-5p mimic, miR-125b-5p inhibitor or control scramble (Qiagen), using the Targefect reagent in the presence of virofect enhancer (Targeting Systems). Transfected hepatocytes were cultured in Hepatocyte Culture Medium (Lonza).
5
Transfection of Primary Hepatocytes
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Freshly isolated primary human hepatocytes were provided by Regenerative Medicine and Experimental Surgery, Hannover Medical School, Hannover, as reported.(12 ) Mouse primary hepatocytes were isolated from mouse liver as described.(8 ) Briefly, mice were anesthetized, and their livers were perfused with Liberase (Roche). After perfusion, livers were disintegrated mechanically before collecting hepatocytes by low‐speed centrifugation. Nonparenchymal cells were removed by discarding the supernatant. For all in vitro transfection experiments, we used Percoll density gradient‐purified mouse hepatocytes to achieve high transfection efficiency. We seeded 100,000 primary hepatocytes per well of a collagen‐coated 12‐well plate (TPP). Twelve hours after seeding, hepatocytes were transfected with 25 nM or 50 nM miR‐125b‐5p mimic, miR‐125b‐5p inhibitor, control scramble (Qiagen), or 100 nM ABTB1 small interfering RNA (siRNA) (Qiagen) using Targefect reagent in the presence of virofect enhancer (Targeting Systems). Transfected hepatocytes were cultured in hepatocyte culture medium (Lonza) containing recombinant epidermal growth factor (an inducer of hepatocyte proliferation), transferrin, ascorbic acid, insulin, hydrocortisone, bovine serum albumin, and gentamicin sulfate‐amphotericin.
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