Qubit 3.0 rna br assay kit

Total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA), phenol-chloroform extraction, and ethanol precipitation followed by purification in a modified protocol from RNeasy Mini Kit (QIAGEN, Germantown, MD). RNA was quantified using either a Qubit 3.0 RNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA) or Nanodrop 2000. Quantification of transcript levels was determined by real-time RT-PCR (iCycler; Bio-Rad Laboratories, Inc, Hercules, CA). qPCR reactions contained 1x final primer concentration (QIAGEN primers, Germantown, MD) or 1 μM sense and 1 μM antisense oligonucleotides (Invitrogen custom DNAoligos, Carlsbad, CA) with SYBR Green (Bio-Rad, Hercules, CA). Primer sets used (QIAGEN QuantiTect) were Hs_PER2_1_SG (QT0011207), Hs_PKM_1_SG (QT00028875), Hs_LDHA_1_SG (QT00001687), Hs_SIRT3_1_SG (QT00091490), Hs_ACTB_2_SG (QT01680476), and Hs_COX4|2_1_SG (QT00044933), Mm_Per2_1_SG (QT00198366), Mm_Angptl4_1_SG (QT00139748), Mm_Cox4i2_1_SG (QT00137844), Mm_Cldn1_1_SG (QT00159278), Mm_Actb_2_SG (QT01136772). Primers for human OPN4 (Invitrogen, Carlsbad, CA, sense 5′-AGT CGC CCC TAC CCC AGC TA-3′ and antisense 5′-CAC AGC TGC TGC CTC CAT GT-3′) were custom designed. Each target sequence was amplified using the following protocol: 95°C for 3 min, 40 cycles of 95°C for 15 s, 55°C for 0.5 min, 72°C for 10 s, 72°C for 1 min and a melt curve protocol.