Quantstudio 7 pro real time pcr system

Total RNA was extracted with TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), and 1 µg RNA was reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). The cDNA was amplified through qRT‒PCR, performed by QuantStudio™ 7 Pro Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using SensiFAST SYBR Hi-ROX Kit (Meridian Bioscience, Cincinnati, OH, USA). The Ct values for each target gene were normalised to the housekeeping gene glyceraldehyde 3’-phosphate dehydrogenase (GAPDH). A list of sequences of primers used in this study is presented in Supplementary Table S1.

mRNA isolation was conducted one of two ways. For primary and iPSC neurons, the Cells-to-CT kit was used for mRNA isolation and cDNA synthesis (TaqMan Fast Advanced Cells-to-CT kit, A35374). For neuroblastoma cells, the Cells-to-CT kit was used or the combination of the RNeasy Plus Micro kit (QIAGEN, 74134) with iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, 1708840) for mRNA isolation and cDNA synthesis. qRT-PCR was then performed using the TaqMan Fast Advanced Master Mix (ThermoFisher Scientific, 4444556) with QuantStudio 7 Pro Real-Time PCR System.
For qRT-PCR, target genes were normalized and multiplexed with HPRT gene expression (neuroblastoma and iPSC neurons) or with ACTB (rat primary neurons). Reference genes used VIC and target genes FAM dye signal.
TaqMan primer list: HPRT1, Hs02800695_m1; SCD, Hs01682761_m1; SCD5, Hs00227692_m1; SNCA, Hs00240906_m1; ACTB, Rn00667869_m1; SCD1, Rn06152614_s1; and SNCA, Rn01425140_m1.

cDNA was obtained from the RNA extracted for each one of the biological replicates of the 13 landraces (4 biological replicates, 2 treatments) of the mRNA-Seq experiment. The reaction was carried out using the ImProm-II™ Reverse Transcription System (Promega) using the manufacturer’s instruction, 1 µl of Oligo(dT)16 (5 µM) (Eurogentec Ltd, Camberley, UK) and 1µl of each RNA sample. Quantification was performed with a Nanodrop 2000 and after that, the cDNA of the 4 biological replicates were pooled in equimolar concentrations. qRT-PCR was performed for the genes TraesCS1B02G384900, TraesCS3B02G409300 and TraesCS4D02G212300 and tubulin using the primers described in Supplementary Data Sheet 7 and the iTaq Universal SYBR Green Supermix (Bio-Rad), adding 200ng of cDNA and 0.1 µM of each primes. The qRT-PCR protocol was set on QuantStudio™ 7 Pro Real-Time PCR System (ThermoFisher) as follows: 95 °C for 4.5 min, 40 cycles of 95°C for 15s and 60°C for 15s. The melting curve was performed by initially heating in a 4.5°C/s ratio up to 95°C and maintaining for 10s reducing the temperature to 3.44°C/s up to 60°C and heating in a 0.15°C/s to 95°C kept for 10s with fluorescence measurement in the last step of the PCR and melting curve. The relative expression between after- and before-heat samples was calculated using the delta-delta Ct method using tubulin as the reference.

Mitotic and interphase cells were separated by mitotic shake-off and RNA was extracted using the Zymo RNA extraction kit according to the manufacturer’s instructions. cDNA was generated using an RNA to cDNA kit (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was mixed with the specified primers and SYBR green PCR master mix (Thermo Fisher Scientific) and 40 cycles of real-time PCR were carried out on a QuantStudio 7 Pro Real-Time PCR System (Thermo Fisher Scientific). We performed 3 biological replicates of each condition, and qPCR was carried out for each of these in 3 technical replicates. Data were analysed using the ddCT method [37 (link)] normalised to the geometric mean of two housekeeping genes: 18S and HPRT1. Statistical analysis was conducted by a paired t-test, and the mitotic enriched samples were compared to the corresponding control. Data were analysed via Prism.

Samples were tested in a duplex RT-qPCR adapted from NAVECA et al. (51 (link)) for the detection of Mayaro and Oropouche viruses. The reactions were performed in a QuantStudio 7 Pro Real-Time PCR System (Thermo Fisher Scientific, Massachusetts, USA), with the following reaction profile: 50°C for 5 minutes, 95°C for 20 seconds, 45 cycles of 95°C for 3 seconds, and 60°C for 30 seconds. The result was considered positive when the cycle threshold was ≤38 for the analyzed virus.

The gene expression of different markers of osteoblastic cells (ALP and OCN) and markers of fibroblast activity (COL1, FN1, and MMP8) were evaluated by RT-PCR. Total RNA was isolated using the Trifast reagent (EuroClone, Pero (MI), Italy), and RNA was quantified on a Nanophotometer NP80 spectrophotometer (Implen NanoPhotometer, Westlake Village, CA, USA) for analysis of RNA integrity, purity, and concentration. Then, the GoTaq^®2 Step RT-qPCR Kit (Promega, Madison, WI, USA) was used to obtain complementary DNA (cDNA), and SYBR Green (GoTaq^® 2 Step RT-qPCR Kit, Promega) was used to perform RT-qPCR according to manufacturer’s instructions. Gene expression was determined using Quant Studio 7 Pro Real-Time PCR System (ThermoFisher, Waltham, MA, USA). The results were normalized to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH for hGFs and to β-actin (β-ACT) for hOBs using the 2^−ΔΔct method. Primer sequences are reported in Table 1.