Viia7 pcr system

Two micrograms of RNA isolated from each sample (CT, ET, 1-MCP, and LOV, collected after 4 months of cold storage, and after 4 months of cold storage + 5 days of shelf life at 20 °C) were transcribed with the SuperScript VILO cDNA Synthesis kit (Invitrogen). RT-qPCR was carried out with the ViiA7 PCR System (ThermoFisher Scientific) according to the thermal profile described elsewhere^{36 (link)}. The 18 elements used in the candidate gene transcription profiling were retrieved from the set of DEGs detected through the RNA-Seq analysis or from available literature^{8 (link),19 (link)}. The set of candidate genes (listed and detailed in Supplementary Table S7) were involved in key pathways for fruit ripening and scald development in pears. Relative gene expression was represented as a mean of normalized expression, taking into account three independent Ct values. The mean-normalized expression value of each sample was calculated using the method described in previous studies^{67 (link)}.

For RT-qPCR, 2 µg of total RNA was reversibly transcribed with the SuperScript VILO cDNA Synthesis kit (Invitrogen). Real Time qPCR was performed with the ViiA7 PCR system (ThermoFisher Scientific) with the following thermal profile: 95 °C 1 m, 40 cycles of 95 °C 1 m, 60 °C 20 s and 95 °C 15 s, with the final step of 60 °C 1 m and 95 °C per 15 s. Twelve genes were retrieved from the set of DEGs detected within the RNA-seq and investigated. Six genes were related to ethylene (ACS, ACO, ERS1, ERS2, ERF1, and ERF2), one was involved in cell wall metabolism (PG), while the rest of the elements were involved in auxin (Aux/IAA) and production of VOCs (AAT, HPL, ADH and LOX) (Supplementary Tab. S5). Relative gene expression was represented as a mean of normalized expression. In order to represent the relative gene expression the mean normalized expression was calculated. The average of three threshold cycle (CT) values independently calculated was computed to obtain the normalized expression value for each sample with the Qgene software^{61 (link)}.

Total RNA was extracted from sorted cells using an RNeasy Plus Micro Kit (QIAGEN) and reverse-transcribed into complementary DNA using SuperScript III reverse transcription and oligo(dT) nucleotides (Thermo Fisher Scientific). Real-time qPCR was performed using SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific) on a ViiA 7 PCR System (Thermo Fisher Scientific). Cytokine expression from qPCR was calculated as gene expression relative to gapdh as ΔCT. Primers for the genes assessed are shown in Supplementary Table 1.

Real-time PCR amplification was performed using 16S universal primers that target the V3–V4 region of the bacterial 16S ribosomal gene. The qPCR step was performed in triplicate on a VIIA 7 PCR system (ThermoFisher, Waltham, MA, USA) with SYBR Green technology. The specificity of all qPCR products was assessed by systematic analysis of a post-PCR dissociation curve performed between 60°C and 95°C. The absolute number of copies of the 16S rRNA gene was determined by comparison with a quantitative standard curve generated by serial dilution of plasmid standards. The total 16S rRNA gene count was normalized by mg of tissue or ml of plasma.

Approximately 125,000 cells per sample were pelleted, washed with 1 × DPBS, and added to a 96-well quantitative polymerase chain reaction (qPCR) plate. Lysis buffer of 150 μL was added to each sample (Invitrogen Pure Link Lysis buffer). RNA purification was carried out as per manufacturer's instructions. A master mix of One-Step Taqman mix (Thermo Fisher) and Thermo Fisher Taqman assays: HIF-1α (Hs00936368_m1), BCL2 (Hs00608023_m1), Cers2 (Hs00604577_m1), Malat1 (Hs00273901_m1), PTEN (Hs02621230_s1), and GAPDH (4326317E) was made. Six microliters of Taqman mix and 4 μL of RNA were added to a 384-well qPCR plate and analyzed using a Thermo Scientific ViiA7 PCR system with a PCR program of 50°C for 15 min, 95°C for 3 min, followed by 40 cycles of 95°C for 5 s and 60°C for 45 s. Gene knockdown was measured using a ΔΔCT method.

Hearts were collected at the appropriate circadian times (CT26, Tcap mRNA expression increasing, or CT38, Tcap mRNA decreasing). ChIP was performed using a Magna ChIP G kit (Millipore, 17–611) according to the manufacturer’s specifications. Briefly, chromatin was sonicated using Sonic Dismembrator Model 100 (Fisher) to yield ∼500 bp fragments that were confirmed by ethidium bromide agarose gel electrophoresis. DNA was quantified using the Nanodrop ND1000 (Thermo-Scientific) and 50 µg was used for immunoprecipitation. For input, 1% of the chromatin was saved prior to immunoprecipitation. Chromatin was pre-cleared for 1 h at 4°C using 20 µl of magnetic beads, then incubated with rabbit polyclonal anti-BMAL1 (Abcam, ab3350), or control rabbit IgG (Millipore, 12–370) antibody overnight at 4°C. DNA was purified using the kit spin columns, and quantified by PCR using the Fast SYBR Green Master Mix (Quanta Bioscience) and VIIA7 PCR system (Applied Biosystems) under the following conditions; 95°C for 10 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 20 sec, using the Tcap ChIP primers (forward 5′-CCCATCACCACCAGTGAGTCT-3′; reverse 5′-GCCCTTTAAATAGCCCCTTCTTC-3′). DNA abundance was quantified as percent input.