Qpcr kits

Total RNA was isolated using RNAbee (AMS Biotech) and reverse transcribed using Superscript III (Invitrogen). Real-time PCR analysis was performed on an Opticon DNA engine (MJ Research Inc.). The dissociation curve was analyzed for each sample. Relative level of the target sequence against the reference sequence was calculated using the ΔΔ cycle threshold method. Primer sequences were as follows: Hprt1 forward, 5′-TCAGTCAACGGGGGACATAAA-3′, Hprt1 reverse 5′-GGGGCTGTACTGCTTAACCAG-3′; Il2 forward, 5′-GTGCCTAGAAGATGAACTTGGA-3′, Il2 R 5′-AAATGTGTTGTCAGAGCCCT-3′, Dicer1 forward, 5′-TATCGCCTTCACTGCCTTCT-3′ Dicer1 reverse, 5′-TTTTCCACCCGAAGTCTAAGTT-3′; spliced Mtor forward 5′-ACCGGCACACATTTGAAGAAG-3′ Mtor reverse, 5′-CTCGTTGAGGATCAGCAAGG-3′; spliced Rictor forward, 5′-ACCGACACCATCACCATGAAG-3′ Rictor reverse, 5′-GACACCATAGACCTAACTGAGGA-3′; unspliced Mtor forward, 5′-TACCGGGTGAGAGATGGGTC-3′ Mtor reverse, 5′-CACAGTGAGCAGGAGAGAG-3′; unspliced Rictor, forward 5′-TGGTGGTAAAAGAGTGGC-3′ Rictor reverse 5′-GTGTAAGTCAGAGGACGG-3′. microRNAs were quantified using miScript RT and qPCR kits according to the manufacturer’s instructions (QIAGEN).