Microamp

The cDNA synthesis from total RNA was prepared using AffinityScript (Stratagene, Agilent Technologies) in a fast eight‐tube strip (0.1 ml, MicroAmp™, Applied Biosystems®, Life Technologies, Paisley, UK) on ice. The total reaction volume of 20 μl contained 0.5 μg RNA, 10 μl cDNA synthesis master mix, 3 μl Oligo dT primer, 1 μl AffinityScript RT RNase block enzyme mixture, and RNAase-DNAse free water (5 Prime GmbH) to 20 μl total volume. Reactions were performed with an initial stage of 25°C for 5 minutes, 42°C for 45 minutes, and 95°C for 5 minutes (Veriti 96 well fast thermal cycler, Applied Biosystems®, Life Technologies). Following synthesis, cDNA was stored in aliquots at –20°C.

Multiplex mRNA primers were custom synthesized (IDT) against human PFKFB1-4 as described previously [20 (link)]. cDNA from normal human tissues and matched tumor and adjacent normal tissues (Clontech, Mountain View, CA) were analyzed using these primers and standard PCR conditions. PFKFB1-4 mRNA expression was determined using real-time RT-PCR with TaqMan probes for human PFKFB1-4 and β-actin (Applied Biosystems, Foster City, CA) in triplicate in 96-well optical plates (MicroAMP®, Applied Biosystems). Analysis of results and fold differences between samples were determined using StepOne software (version2.1) (Applied Biosystems) and calculated from the ΔΔCT values with the formula (2^−ΔΔCT). The data are represented as the mean ± SD from triplicate measurements from three independent experiments. For calculation of copy number, the molecular weight was determined for the double stranded DNA sequences of the 4 PFKFB isoforms. The OD of the DNA (in gm/μL) was divided by the molecular weight of the product (result in moles/μL) and then this number was multiplied by Avogadro's number (6.022 × 10²³ molecules/mole). The resulting number of DNA molecules per μL was used to generate a standard curve from which copy numbers were calculated. Statistical significance was assessed by the two-sample t test (independent variable).

Reverse Transcription: cDNA was synthesised by using Bioline Sensifast cDNA synthesis kit (Bio-65053). Taqman® oligonucleotide probes have been used in the qPCR experiments (Table 1) Real-time qPCR was done using the Bioline SensiFAST Probe Hi-ROX Kit (BIO-82002). The target genes included human β, γ globin. The qPCR reactions were performed in triplicates in 96- well optical plates (Microamp®, Applied Biosystems, N8010560).
The reactions were run on the Applied Biosystems Step-One Plus Real-Time PCR system machine. Results were analysed using the Life Technologies Step-One software.

Respiration rate was measured in HM discs by sealing each of the wells in the 24 well plate with an adhesive PCR plate cover (MicroAmp, Applied Biosystems, Foster City, California, USA) and attaching an adhesive septum (Bridge Analyzers Inc. Alameda, California, USA) to the top. Samples (1 mL) were collected by inserting a syringe into the airspace above the discs. Respiration rate was measured 1, 2, and 3 days after harvest. Wells were sealed for 5–11 min. CO₂ production (μl CO₂ min⁻¹ g⁻¹ fresh weight) was measured using an infrared CO₂ analyzer (Horiba, Irvine, CA).

Primers were designed based on the sequence of 21 contigs showing in silico expression levels stable or variable among experimental modalities, and checked with NetPrimer (Premier Biosoft, Palo Alto, California). Amplification specificity and qPCR efficacy were checked for each contig as described [81 (link)]. Primer pairs retained for contig expression measurement had an efficiency value between 80 and 110 % (Additional file 3: Table S2). The expression level of the 21 contigs was measured in each of the 42 individual RNA samples used to create the 14 pooled samples subjected to RNA-Seq. Analyses were performed in duplicate (technical replicates) for each sample. Reverse transcription was performed using the Masterscript RT-PCR System kit (5 PRIME, Hamburg, Germany) starting from 5 μg total RNA. The StepOnePlus™ Real-Time PCR System Thermal Cycling Block (Applied Biosystems, Foster City, USA) was used to perform qPCRs in fast optical 0.1 mL, 96-well reaction plates (MicroAmp™, Applied Biosystems, Cheshire, UK). Reaction mixes, PCR programs, contig expression measurement and normalization with three previously validated reference genes using a five-point standard dilution curve were as described [81 (link)].

cDNA was generated using a High Capacity Reverse Transcription kit (Applied Biosystems). Quantitative PCR reactions were performed in MicroAmp optical 384-well reaction plates and analyzed using a QuantStudio 5 PCR system (Applied Biosystems). Reactions were performed in triplicate or quadruplicate and included primers for β-actin (ACTB) as a housekeeping gene. Primers were designed using the Universal Probe Library (UPL) Assay Design Center (Roche) and purchased from Integrated DNA Technologies. Raw fluorescence data were converted to Ct values using the Thermo Fisher Cloud facility and normalized to ACTB. For primer sequences and associated probes see Supplemental Table 5.