Mouse hprt primepcr probe assay

Murine tissues were collected into Trizol reagent (ThermoFisher), homogenized and immediately flash-frozen until ready for total RNA isolation. RNA was subsequently digested for 20 minutes at 37 °C with recombinant DNase I (ThermoFisher), then analyzed for integrity and concentration on an Agilent nucleic acid bioanalyzer (Agilent Technologies). Synthesis of cDNA used 1 μg of RNA, which was reverse transcribed using the iScript cDNA Synthesis kit (BioRad) according to the manufacturer’s protocol. For each transcript assay, droplets were generated using 50 ng of cDNA, 900 nM primers, 250 nM probes in 1x ddPCR Supermix for Probes (BioRad) on a QX200 Droplet Generator, followed by PCR amplification. The sequence of primers and probes used are listed in the appended table. PCR cycling conditions consisted of an initial enzyme activation step for 10 minutes at 95 °C, followed by 40 cycles of 94 °C for 30 seconds and 59 °C for 30 seconds with a 2 °C/second ramp rate, and a 10 minute enzyme deactivation step at 98 °C for 10 minutes. Each reaction was duplexed with the Mouse Hprt PrimePCR Probe Assay (assay ID #qMmuCEP0054164, HEX, BioRad) and performed in triplicate. Upon completion of reactions, samples were read on a QX200 droplet reader (BioRad) to obtain expression levels relative to murine Hprt and transcript-specific copy number, then further analyzed using Excel software.