Qpcrbio probe mix no rox

Levels of mRNA were measured exactly as described previously^{38 (link)}. Briefly, interscapular BAT was isolated in Invitrogen TRI Reagent, lysed and homogenized with QuickPrep Adaptor (Fisher Scientific). RNA isolation was performed according to the manufacturer (Fisher Scientific) and cDNA synthesized using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific), Invitrogen RNaseOUT Recombinant Ribonuclease Inhibitor (Fisher Scientific) and T100 Thermal cycler (Bio-Rad). Gene expression of cDNA (30 ng) was determined using the qPCRBIO Probe Mix No-ROX (PCR Biosystems), performed on the MyiQ Single-Colour Real-Time PCR Detection System (Bio-Rad). Samples were measured in duplicates and the 2^−∆∆Ct method employed to determine fold change in gene expression. The target genes were normalized to the housekeeping gene, peptidylprolyl isomerase A (PPIA). All Applied Biosystems TaqMan Gene Expression Assay (FAM-MGB) (Major Resources Table) were purchased from Fisher Scientific.

For ABI thermal cyclers, each 10-μL qPCR contained final concentrations of 1 × TaqMan Universal Mastermix II, with Uracil-DNA N-glycosylase (UNG, a common method to minimize PCR cross-contamination by enzymatic degradation of previous PCR products with incorporated dUTP; Life technologies, Paisley, UK), each oligonucleotide at 0.3 μM and 2 μL template DNA in aqueous solution. In the UK, the assay was performed on an ABI 7900HT Fast Real Time PCR machine (Life Technologies, Paisley, UK). In Tanzania, the assay was performed on an Applied Biosystems ViiA 7 Real Time PCR machine (Life Technologies, Paisley, UK). Both instruments utilized a 384-well format.
For Rotor-Gene thermal cyclers samples were run in a 72-well rotor format on a Corbett Rotor-Gene 3000. Each 20-μL qPCR contained 1 × qPCRBIO Probe Mix No-Rox (PCR Biosystems, London, UK), each oligonucleotide at 0.3 μM and 8 μL template DNA in aqueous solution.
No-template controls and serial dilutions of known-concentration PCR product were included on each run on all systems. Thermal cycling conditions for all systems were 50 °C for 2 min, 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 60 s. Samples with quantitation cycle (C_q) values < 15 cycles were diluted and retested.

To ensure sample quality without erythrocyte miRNAs contamination, the level of haemolysis in all serum samples was assessed by spectrophotometry (NanoDrop 1000, USA) [11 (link)]. No serum sample had A414 reading above a value of 0.2 [13 (link)].
Extracellular RNA was isolated from 300 μL of serum by using miRCURY™ RNA isolation kit for biofluids (Exiqon, Denmark). PCR conditions and all reaction mixes were adopted from the user bulletin by Applied Biosystems (Protocol for Creating Custom RT and Preamplification Pools using TaqMan® MicroRNA Assays).
Briefly, multiplex reverse transcription (RT) was performed with TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, CA, USA) and RT primer pool prepared by mixing 5x RT primers provided in TaqMan MicroRNA Assays (Applied Biosystems, CA, USA). To preamplify all tested miRNAs together, TaqMan PreAmp Master Mix was used with PreAmp Primer Pool prepared from 20x TaqMan MicroRNA Assay (Applied Biosystems, CA, USA). qPCRBIO Probe Mix No-ROX (PCR Biosystems, United Kingdom) and 20x TaqMan MicroRNA Assays (Applied Biosystems, CA, USA) were used to perform RT-PCR of individual miRNAs (RotorGene3000 system Corbett Research, Sydney, Australia). All TaqMan MicroRNA Assays are listed in Supplementary material (Online Resource 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/1246129).