Taqman reverse transcription and universal pcr master mix reagents

RT-QPCR was used to validate gene expression levels of selected genes using TaqMan® chemistry. Probes and primer sets specific for detection of mouse RNA transcripts were purchased from Applied Biosystems, Foster City, CA. These included gene-specific probes for the following mouse genes: Igk-V28, assay ID# Mm01742005_g1; Ccr9, assay ID# Mm02620030_s1; Foxp3, assay ID# Mm00475162_m1; Lta, assay ID# Mm00440228_gH; Cdk6, assay ID# Mm01311342_m1; Jun, assay ID# Mm00495062_s1; Nov, assay ID# Mm00456855_m1. Gene-specific probes labeled with FAM (6-carboxyfluorescein) in the 5′ end, and with a dark quencher in the 3′ end were used for all the target genes of interest. For each sample, GAPDH was used as the endogenous control gene (assay ID# Mm99999915_g1) using a mouse-specific probe labeled with FAM (6-carboxyfluorescein) in the 5′ end, and with a dark quencher in the 3′ end. The experiments were performed in the ABI Prism 7500 Sequence Detection System (Applied Biosystems) using the TaqMan® Reverse Transcription and Universal PCR Master Mix Reagents. All the samples were tested in triplicate. The cycling conditions were 48°C for 30 min, 95°C for 10 min, 40 cycles of 95 °C for 15 s, and 60°C for 1 min. The 2−ΔΔCt method was used to calculate fold changes in the expression levels of the genes of interest(8 (link)).