Express sybr greener qpcr supermix

xanthine oxidase from bovine milk, luminol sodium salt, xanthine, oxypurinol, PBS tabs, Na-EDTA salt, gelatin from bovine skin, penicillin/streptomycin, trypsin-EDTA, Trolox, and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium perborate, boric acid, NaOH, and FeCl₂ were from Carlo Erba (Milan, Italy). M200 medium, low serum growth supplements, and fetal bovine serum, RNaseOUT, were purchased from Thermo Fisher Scientific (Waltham, MA, USA). RNeasy Mini Kit was from QIAGEN (Hilden, Germany). Primers for RT-PCR were purchased from IDT (Coralville, IA, USA). Cell counting kit-8 (CCK8) and LDH assay kit were purchased from Dojindo Molecular Technologies (Rockville, MD, USA). SuperScript® III First-Strand Synthesis SuperMix and EXPRESS SYBR® GreenER™ qPCR SuperMix were purchased from Life Technologies (Carlsbad, CA, USA). All the other chemicals and solvents were of the highest analytical grade.

qRT-PCR was performed to validate target gene expression as previously described42 . In brief, total RNA was extracted and cDNA synthesis was performed with a SuperScript VILO cDNA Synthesis kit (Life Technologies, 11754050). Diluted cDNA was used for PCR reaction with Express SYBR GreenER qPCR Supermix (Life Technologies, 11784-200) according to manufacturer’s instruction. The quantitative analysis was performed by Relative Expression Software Tool (REST) 200948 (link). Expression values were normalised with 2 reference genes including 18SrRNA and GAPDH.

Brains were collected from pollen foragers, and their dorsal regions (including calyces and peduncle) dissected quickly, frozen on dry ice and stored at –80°C until RNA extraction, then homogenized individually in Trizol. RNA was isolated using RNA easy Mini purification kit (Qiagen) and RNA concentrations were measured using Quant-iT Ribo Green RNA assay (Invitrogen).
Real-time quantitative PCR (qPCR) analysis was performed as previously described [59 (link)]. 50 ng of RNA was reverse-transcribed using VILO Supercript (Invitrogen). Gene-specific amplification products were generated using ExpressSYBR® GreenER qPCR SuperMix (Invitrogen) and gene specific primer pairs (S2 Table). Assay efficiencies were derived from standard curves generated using pooled cDNA from all the bees. We used the ΔΔCt method with assay efficiencies incorporated in the formula to calculate transcript abundances: Normalized = (1 + Efficiency target, ^{- ΔCt target}) / (1 + Efficiency reference, ^{- ΔCt reference}). Transcript levels were normalized using the geometric mean of two reference genes, Am18S and elongation factor-1 alpha elf1α, as they show the smallest variation of all potential housekeepers tested (NormFinder).