Mx3000p multiplex quantitative pcr system

Total RNA was isolated from 2×10⁵ cells using the NucleoSpin RNA II kit, according to the instructions of the manufacturer (Macherey-Nagel, Düren, Germany). Reverse transcription was performed from 1 μg of total RNA by using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific). Synthetic primers for HS3ST3A, HS3ST3B and HS3ST4 were described in [24 (link)]. Synthetic primers for HS3ST2 were designed by using Primer-Blast (NCBI): 5’-GCTCTCGAGGGTCCTGGGCA-3’ (forward), 5’-TGTGGGCGTGAAGAAGGGGG-3’ (reverse). Specificity of the primers was checked by semi-quantitative RT-PCR on a 2.5% (w/v) agarose gel. All of them amplified only one fragment of expected size, for which the sequence was confirmed (GATC Biotech, Constance, Germany). Real-time PCR amplifications were performed using an Mx3000P Multiplex Quantitative PCR system (Agilent Technologies, Santa Clara, CA, USA), as described in [26 (link)]. The transcript of HPRT was used as a control to normalize the expression of our genes of interest. The amplification efficiency of each primer pair was performed on serial dilutions of cDNA. The point at which the PCR product was first detected above a fixed threshold, termed cycle threshold (Ct), was determined for each sample, and the average Ct of triplicate samples was used for analysis.

qPCR experiments were performed on the Mx3000P Multiplex Quantitative PCR System (Agilent Technology, La Jolla, CA, USA) using as template 1 μL of the cDNA, synthesized from total RNA according to the Invitrogen SuperScript II RNase-reverse transcriptase manual (Life Technologies Corporation, Carlsbad, CA, USA) and Brilliant SYBR^® Green QPCR Master Mix (Agilent Technologies, La Jolla, CA, USA). Cycling conditions were: 10 min at 95 °C for activation of the Hot start Taq polymerase and 40 cycles for the melting (30 s at 95 °C), annealing (30 s at 61 °C), and extension (30 s at 72 °C). Each qPCR measurement was carried out in triplicate using specific primers for either ShBLE or APHVIII (Table A1). The UBC8 gene, encoding a ubiquitin ligase polypeptide (XM_001697453), whose expression was previously shown to be constitutive under the different conditions used [52 (link),53 (link)] and was used as a housekeeping gene to normalize mRNA abundance. 2^−ΔΔCT approach was used to calculate fold change relative to the expression level of a reference clone, which was chosen among the transformants which showed the lowest values [54 (link)].