ADH2 gene expression was determined by real-time PCR. An efficiency test was performed using cDNA from S. cerevisiae and L. fermentati as the template. The PCR efficiency was scaled to 95–105% and above, 0.999 R-squared and a slope of −3.33, as recommended (Bustin et al., 2009 (link)). Real-time PCR assays were performed in an iCycler (iQ™5, BioRad, USA), using the standard thermal cycling protocol. The reaction mixture of 20 µl contained 10 µl of iTaq Universal SYBR® Green Supermix (2x ) (BioRad, USA), 0.75 nM of forward and reverse primers, 2 µl of 10-fold diluted cDNA, and deionized water to reach the final volume. Real-time PCR experiments were carried out in two replicates, of which each real-time PCR was performed in duplicate on each sample. A negative control without the cDNA template was included. The thermal amplification program used was as follows: 95 °C for 30 s; 40 cycles of 95 °C for 10 s; and 60 °C for 30 s before the melt-curve was collected from 58 °C to 90 °C. Figure S1 displayed melt curve analysis for ADH2 and Act1 genes during the amplification. Gene expression levels were shown as the threshold cycle (Ct) of the studied gene normalized with the Ct of a reference gene, ACT1 (Table S1 ).
Itaq universal sybr green supermix 2x
Manufactured by Bio-Rad
Sourced in United States
ITaq Universal SYBR Green Supermix (2X) is a ready-to-use real-time PCR reagent that contains all the necessary components, including SYBR Green I dye, for the detection and quantification of DNA targets. It is formulated for use with a wide range of thermal cycler platforms.
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Lab products found in correlation
Cfx96 real time pcr system, by Bio-Rad (3 mentions)
Cfx connect real time pcr system, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Cfx connect qpcr machine, by Bio-Rad (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Biophotometer plus, by Eppendorf (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cfx96 c1000 touch thermal cycler, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
9 protocols using itaq universal sybr green supermix 2x
1
Quantitative Analysis of ADH2 Expression
2
Quantitative PCR Gene Expression Analysis
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The qPCR reaction was carried out by CFX ConnectTM Real-Time PCR System (BIO-RAD, Hercules, CA, USA) using the iTaq Universal SYBR Green supermix 2X (BIO-RAD, USA) and specific primers for individual gene (Table 2 ). The qPCR was performed in triplicate using 100 ng of cDNA, 400 mM of primers. Thermal cycling conditions were 95 °C for 30 s (holding stage); 40 cycles of 95 °C for 15 s, and 60 °C for 30 s (cycling stage); followed by 95 °C for 15 s; 60 °C for 60 s; and 95 °C for 15 s (melt curve stage). Changes in the expression levels of the above genes were measured using the 2−ΔΔCt method and a standard curve [56 (link)].
3
Quantifying Gene Expression with qPCR
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One hundred fifty nanograms of RNA was used to convert to cDNA using the Applied Biosystems High‐Capacity cDNA Reverse Transcription Kit following the manufacturer's instructions (Cat # 4368814). The cDNA thus obtained was diluted at 1:10, 1:100, and 1:1000. The housekeeping gene, 18 s, was run at all three dilutions to ensure the quality of the cDNA. Five microlitres of the 1:100 cDNA reaction was used to perform qPCR on the gene of interest. For each qPCR reaction, the 5 μL cDNA was added to 10 μL of iTaq Universal SYBR Green Supermix (2X) (Biorad Cat #1725120), 1.5 μL of each primer (375 nM concentration), and 2 μL of nuclease free water. Thus, a total volume of 20 μL of qPCR mix was run on the BioRad CFX connect qPCR machine. The qPCR data for each gene were analysed using the double delta Ct method.
4
Total RNA Extraction and qPCR Analysis
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Total RNA was extracted using the SV Total RNA isolation system (Promega Z3100 (Promega, USA)), followed by DNase treatment to eliminate DNA contamination. The integrity of the RNA in all samples was verified by comparing the ribosomal RNA bands in ethidium bromide-stained gels. RNA sample purity was estimated using spectrophotometric measurements at 260 and 280 nm (Eppendorf Biophotometer plus (Eppendorf, Germany)). The OD260/280 of all samples was 1.8–2.2. Real-time PCR reactions were performed in a 20 µl total reaction volume comprised of 10 µl of iTaq universal SYBR Green supermix(2x) (Bio-Rad Laboratories Inc., USA), 1 µl of each gene specific primer (Table 1 ), 2 µl of cDNA templates, and 6 µl of PCR-grade water. Reactions were carried out on a CFX-96 real-time PCR system (Bio-Rad Laboratories Inc., USA). The efficiencies of the target and reference genes were similar [5] , [21] . The PCR parameters for all genes were as follows: 95°C for 3 min, 40 cycles of 95°C for 5 sec, 30 sec at the Tm value of primer pairs (Table 1 ) with melting curve analysis performed to determine the specificity of PCR products. Every treatment included four replicates, and each reaction was run in triplicate.
5
Real-Time PCR Amplification Optimization
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Real-time PCR was performed on a CFX Connect™ Real-Time PCR System by using iTaq Universal SYBR Green Supermix (2X) (Bio–Rad Laboratories, CA). DNA (25 ng) was performed in triplicate along with a control for each test. Amplification was performed by an optimized protocol (5 min at 95 °C, 40 repeated cycles of two steps at 95 °C for 15 s, 55 °C X gene/56 °C Y gene for 15 s and 72 °C for 30 s).
6
Quantitative RT-PCR for Gene Expression
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Total RNA was extracted with Trizol (Invitrogen, Monza, Italy) and 1 μg was converted into cDNA using SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen), according to the manufacturer’s instructions. RT–PCR was performed with 50 ng of cDNA template in a 25-μL total reaction volume (12.5 μL Bio-Rad iTaq Universal SYBR Green supermix (2X), 0.5 mM of each gene-specific primer, H2O up to volume). Reactions were carried out on a Bio-Rad CFX-96 real-time PCR system (Bio-Rad Laboratories, Segrate, Italy). Each reaction was run in triplicates. For amplification, the following primers were used: STAT5, forward (5′-CTGAACAACTGCTGCGTCAT-3′), and reverse (5′-GTGGACGATGACAACCACAG-3′); GAPDH, forward (5′-GGAGTCAACGGATTTGGTCGT-3′) and reverse (5′-GCTTCCCGTTCTCAGCCTTGA-3′); BCR-ABL, forward (5′-CCACTGGATTTAAGCAGAGTTCAA-3′), and reverse (5′-TCCAACGAGCGGCTTCAC-3′).
7
Molecular Analysis of H. pylori Antibiotic Resistance
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The antrum and fundal samples were extracted separately, and only positive samples were processed. For analysis, the 23S ribosomal RNA (23S) and the oxygen-insensitive NADPH nitroreductase (rdxA) regions were sequenced for clarithromycin and metronidazole resistance, respectively (Table 1 ). First, the glmM, which encodes for a phosphoglucosamine mutase, was used to confirm that samples were PCR-positive for H. pylori. All PCR mixtures consisted of 10 µL of BioRad iTaq Universal SYBR Green Supermix (2X); 200 nM of each forward and reverse primer and one microliter of H. pylori DNA (20–50 ng) were added to each reaction mixture, with a final volume of 40 µL. Antibiotic-resistance-associated genes were run in a BioRad CFX96 real-time PCR system. The PCR programs for amplification consisted of 5 min at 95 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 55–63 °C, 40 s at 72 °C, and a final incubation at 72 °C for 3 min. In some cases, PCR products (7 µL of each sample) were electrophoresed in 2% agarose gels stained with SYBR Green for 1 h at 100 V.
8
Characterization of Parathyroid and Thyroid Markers in Organoid Cultures
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Total RNA isolation was performed from the organoid cultures using a NucleoSpin RNA XS isolation kit (Macharey-Nagel®, Germany) after 14 and 30 days of incubation. The manufacturer’s instructions were followed. Purity was monitored using a NanoDrop spectrophotometer (Thermo Scientific®). A reverse transcription reaction was performed using the iScript cDNA Synthesis Kit (Bio-Rad®) using a thermal cycler according to the manufacturer’s description. Gene expression profiles of the selected parathyroid-specific markers PTH, CaSR, and PTH1R, thyroid-specific marker Thyroid peroxidase (TPO) were investigated. Real-time PCR was carried out using iTaq Universal SYBR Green Supermix (2X) (Bio-Rad®). The reaction setup was prepared based on the manufacturer’s instructions. β-actin gene was employed for normalization and internal control purposes. The reaction was performed with Bio-Rad CFX96 C1000 Touch thermal cycler (Bio-Rad®). Data analysis was carried out by the comparative Ct method and data were expressed as fold change. Primer sequences and amplicon lengths are shown in Table 1 .
9
RT-qPCR for Autophagy and Apoptosis
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The RT-qPCR was conducted using a CFX96 real-time PCR detection system (Bio-Rad, USA) with iTaq Universal SYBR Green Supermix (Bio-Rad, USA). The reaction mixture consisted of 2 μl of cDNA template, 10 μL of iTaq Universal SYBR Green Supermix (2X) (Bio-Rad, USA), and 0.3 μM of each forward and reverse primer (Table 2 ), adjusted to a final volume of 20 μl with deionized water. The thermal cycling conditions included pre-incubation at 95°C for 30 seconds, followed by 40 cycles of amplification at 95°C for 5 seconds and 60°C for 30 seconds. The final melting step involved a gradient from 65°C to 95°C with a ramping rate of 0.5°C/5 seconds. Relative gene expression levels for autophagy and apoptosis were calculated using the 2-ΔΔCt method, where ΔCt represents the differences in the cycle threshold number between the target gene and β-actin, and ΔΔCt represents the relative change in differences between the wild-type (WT) and mutant groups.
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