Stepone plus thermal cycler system

Total RNA (10 ng) was reverse transcribed and subjected to TaqMan miRNA assays (Applied Biosystems, ThermoFisher Scientific, Foster City, CA) in a StepOne plus thermal cycler system (Applied Biosystems). Briefly, total RNA was combined with RT Master Mix and gently-mixed. Next, 5X RT primer for miRNA assay was added into the RT reaction tube, mixed, and incubated on ice for 5 min. RT reaction protocol for thermal cycler was as follows: 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C in a Veriti thermal cycler (Applied Biosystems). For the qPCR analysis, the cDNA was added to the reaction mix containing TaqMan^® Universal Master Mix II, with UNG, 20X TaqMan^® MicroRNA Assay (primer), and nuclease-free water, and mixed. qPCR reaction conditions were followed as per manufacturer instruction. U44 RNA was used as an internal control. Relative miRNA expression was calculated by the ΔΔCt-method using the StepOne Software version 2.1 from Applied Biosystems.

Total RNAs for miRNA microarray were isolated from enriched TECs (based on our previously published enzymatic method [27 (link)]) of young and aged murine thymuses using QIAGEN miRNeasy Mini Kit (QIAGEN Cat#217004) according to the manufacturer’s instructions. The miRNA microarray assay was performed through a service by LC Sciences, LLC (www.lcsciences.com) with miRBase-V20 arrays containing 1892 unique probes. Total RNAs for real-time RT-PCRs from enriched TECs of freshly isolated murine thymuses or from TEC line Z210 were isolated with TRIzol reagent (Invitrogen) and reverse transcribed to cDNA with the SuperScriptIII cDNA kit (Invitrogen). Real-time RT-PCRs for miRNAs were performed with TaqMan® MicroRNA Assay kits of individual miRNA primers and probes (Applied Biosystems). Real-time RT-PCR of FoxN1 primers and probe (TaqMan method) were as described previously [27 (link)]. Real-time RT-PCR was run on a Step-One-Plus thermal cycler system (Applied Biosystems). The microRNA results were internally normalized to U6snRNA control levels, while the relative expression levels of FoxN1 mRNAs from the Z210 were internally normalized to GAPDH levels. The average ΔΔC_T value from multiple young animals was always arbitrarily set as 1.0 in each real-time PCR reaction.