MiRNA mimics (miR-146a, Cat. 4464066 ID “MC10722”; miR-155, Cat. 4464066 ID “MC28440”; and control Cat. 4464058) were obtained from Applied Biosystems. OFs were grown to 70% confluence in six-well plates and treated with miRNA mimics mixed with RNAiMAX in Opti-MEM I at 100 nM for 24 hours. Cells were incubated in DMEM containing 0.1% FBS for a further 24 hours before harvest.
Mir 155
Manufactured by Thermo Fisher Scientific
MiR-155 is a microRNA (miRNA) primer and probe set for detecting and quantifying the expression of the miR-155 gene. MiR-155 is a small non-coding RNA molecule that plays a role in the regulation of gene expression. The MiR-155 primer and probe set can be used in real-time PCR assays to measure the levels of miR-155 in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Mir 146a, by Thermo Fisher Scientific (3 mentions)
Mir 21, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Mir 200c, by Thermo Fisher Scientific (1 mentions)
Mir 29a, by Thermo Fisher Scientific (1 mentions)
Mir 125a 5p, by Thermo Fisher Scientific (1 mentions)
Lc480 light cycler, by Roche (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Mir 17, by Thermo Fisher Scientific (1 mentions)
Mir 19b, by Thermo Fisher Scientific (1 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rnu6b, by Thermo Fisher Scientific (1 mentions)
Mirvana paris total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Mir 210, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using mir 155
1
miRNA Mimics Transfection in OFs
2
Quantification of Mature miRNA and mRNA Expression
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Mature miRNA expression was quantified using TaqMan microRNA assays (ABI, Life Technologies, Carlsbad, CA, USA). Total RNA (5 ng) was reverse-transcribed using miRNA specific primers and the TaqMan Reverse Transcription Kit (ABI, Life Technologies, Carlsbad, CA, USA). TaqMan miRNA assays were performed on a LC480 LightCycler (Roche, Basel, Switzerland), using the TaqMan Universal PCR Master Mix (ΑΒΙ, Life Technologies, Carlsbad, CA, USA), and analyzed with the LC480 analysis software. Values were normalized to the endogenous control sno202. For quantification of IFN-γ mRNA expression, 10 ng RNA was transcribed using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Expression of IFN-γ was analyzed using TaqMan gene expression assays and the data was normalized to GAPDH endogenous control. Relative fold changes of miRNA or mRNA expression were determined with the 2−ΔΔCT method [18 (link)]. Mmu-miRNA and mRNA detection assays were obtained from Applied Biosystems: sno202 (assay ID: TM 001232); miR-125a-5p (assay ID: TM 002198); miR-155 (assay ID: TM 002571); miR-200c (assay ID: TM 002300); miR-29a (assay ID: TM002112); IFN-γ mRNA (assay ID: Mm 01168134_m1); and GAPDH (assay ID: Mm 99999915_g1).
3
Quantifying miRNA Expression in Immune Cells
4
Validating miRNA Expression via qPCR
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To confirm the results from miRNA-Seq, we selected some miRNAs of interest to measure using PCR. Total RNA was isolated from AMSCs by the mirVana PARIS total RNA isolation kit (Life Technologies, Carlsbad, CA, USA, Cat# AM1556) according to the kit protocol. RNA concentrations were measured by a NanoDrop Spectrophotometer (NanoDrop, Thermo Fisher Scientific, Inc.). A fixed volume of 5 μL of RNA elute at 1 ng/uL was reverse transcribed by using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Cat# 4366596). For PCR, 1.33 μL of RT product was combined with 10 μL of TaqMan Universal Master Mix (Cat# 4440038), 7.67 μL of H2O and 1 μL of primers, including miR-21, miR-146a, miR-155, and miR-210 (Life Technologies, Cat# 000397, 001097, 002623, and 000512 respectively) to make up a 20 μL reaction. RNU6B (Life Technology Cat# 001093) was included in the assay as reference control. Real-time PCR was carried out on an Applied Biosystems (Foster City, CA, USA) ViiA7 Real-Time PCR system at 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Fold changes of miRNA levels in hypoxia relative to normoxia were calculated using the 2−ΔΔCt method.
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