Nucleic acid extraction from the samples was performed with the RINA™M14-01 nucleic acid extraction device using the extraction kit RN-NA-14-111-100. PCRs were achieved with “Bio-Speedy® COVID-19 RT-qPCR Detection Kit (Cat No BS-SY-WCOR-305, Bioeksen R&D Ltd., Turkey)” and Bio-Rad CFX96 Touch™ (Bio-Rad Laboratories, Inc. USA). For the evaluation of results, replication curves of the FAM/HEX channels were observed. Human RNase P was used as an internal control to rule out false-negative results for each sample. Also, one positive patient’s sample with a known ct value was used as an internal quality positive control. LoD of Bio-Speedy® COVID-19 RT-qPCR Detection Kit is 20 copy/ml. Non-sigmoidal curves were recorded as negative. In the cases where positive, negative, and internal control values met the appropriate criteria, Ct < 40 was assumed positive. Note that patients were assumed positive either through positivity of at least two separate clinical samples or at least two positive results obtained from the same clinical sample according to WHO recommendations [9 ]. SARS-CoV-2 viral load analysis were performed with RdRp (RNA-dependent RNA polymerase) gene targeted Bio-Speedy® SARS-CoV-2 RT-qPCR Kit (BS-SY-WCOR-307) (Bioeksen, Turkey) and Bio-Rad CFX96 Touch™ (Bio-Rad, USA). Viral load of standards synthetic SARS-CoV-2 RdRp fragment/mL was between 2.5 × 102–5 copy/ml.
Cfx96 touch
Manufactured by Bio-Rad
Sourced in United States, Japan, Singapore, China, Canada, Germany, Switzerland, Italy, United Kingdom
The CFX96 Touch is a real-time PCR detection system designed for amplification and quantification of DNA and RNA samples. It features a 96-well format and is capable of performing a variety of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (42 mentions)
Iscript cdna synthesis kit, by Bio-Rad (20 mentions)
Rneasy mini kit, by Qiagen (16 mentions)
Trizol, by Thermo Fisher Scientific (15 mentions)
Itaq universal sybr green supermix, by Bio-Rad (15 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (12 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (12 mentions)
Primescript rt reagent kit, by Takara Bio (10 mentions)
Iq sybr green supermix, by Bio-Rad (10 mentions)
Sybr premix ex taq, by Takara Bio (8 mentions)
Cfx manager 3, by Bio-Rad (7 mentions)
Sybr green master mix, by Bio-Rad (6 mentions)
Mirneasy mini kit, by Qiagen (6 mentions)
Hiscript 2 q rt supermix, by Vazyme (6 mentions)
Cfx manager software, by Bio-Rad (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (5 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Takara Bio (5 mentions)
Hifi mmlv cdna kit, by CoWin Biotech (5 mentions)
Ssoadvanced sybr green master mix, by Bio-Rad (5 mentions)
391 protocols using cfx96 touch
1
SARS-CoV-2 Detection via RT-qPCR
2
Quantifying Gene Expression in Mouse Liver
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Total RNA was extracted from mouse liver tissue and cells by the TRIzol method and reversely transcribed into cDNA. Real-time PCR was performed in the CFX96 Touch™ instrument (CFX96 Touch™, Bio-Rad, Hercules, CA, USA). The reaction conditions were as follows: pre-denaturation was performed at 95 °C for 30 s, one cycle; denaturation at 95 °C, 5 s, 40 cycles; annealing extension at 60 °C, 30 s, 40 cycles. The relative expression levels of target genes were calculated by the 2−ΔΔCT method according to the amplified CT values [52 (link)]. Primer sequences are shown in Table 2 .
3
Quantitative Gene Expression Analysis
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Three replicates of 50 eye imaginal discs of third instar larvae were collected for each genotype. Homogenization was performed with a 1-ml syringe with a 27-G needle. Total RNA was isolated using an RNeasy Lipid Tissue Mini-Kit (Qiagen), followed by a DNAse treatment (DNAse I, Roche), and 0.5 µg of total RNA was reverse transcribed to cDNA using a Prime Script RT reagent kit (TaKaRa) according to the manufacturer’s instructions. RT-PCR was performed in triplicate for each single extraction with SYBR Green Master Mix: SYBR Premix Ex Taq II (Tli RNase H Plus) (TaKaRa) using the CFX96 Touch™ Real-Time PCR Detection System (BioRad), by the specific primer pairs listed in Supplementary Table S1 . Samples were run in triplicate with SYBR® Premix Ex TaqTM II (TaKaRa) using CFX96 touchTM (Biorad), and data were analyzed with a standard curve-based method calculated with CFX ManagerTM software. The specificity of primers was tested with melt curves created by CFX ManagerTM software and the agarose gel electrophoresis of amplified fragments. dRP49 was used as an internal control.
4
RNA Extraction and Real-Time qPCR Analysis
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The RNAs were extracted from 1 × 106 cells using the NucleoSpin® miRNA kit (Macherey-Nagel #740971) in accordance with the manufacturer’s recommendations. Total RNA (500 ng) was reverse transcribed with the iScript cDNA Synthesis Kit® (Bio-Rad #1708890). The cDNAs were diluted to 1/10th in RNase-free water. Real-time PCR was performed with SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad #1725272) and 250 nM of forward and reverse primers specific to the genes of interest, i.e., E-cadherin (cdh1), N-cadherin (cdh2), Vimentin (vim), Fibronectin (fn1), and to reference genes, i.e., β–actin (actb) and GAPDH (GAPDH) (Eurogentec, sequences of primers are available on request) in a CFX96 TouchTM (Bio-Rad). Amplification was performed as follows: 5 min at 95 °C, 35 cycles (15 s at 95 °C and 60 s at 60 °C). PCR results were analysed using the 2−ΔΔCt method.
5
Virus RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from virus-infected plants using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For virus expression analysis, cDNA was synthesized using reverse transcriptase and oligo(dT) primer (TaKaRa, Japan). qRT-PCR assays were carried out in a CFX96 Touch TM real-time PCR detection system (Bio-Rad, Hercules, CA) using SYBR Premix Ex Taq TM II (TaKaRa). Three technical replicates were performed for each biological replicate. The potato TUBULIN2 gene was used as a reference. Primer sequences for qRT-PCR are listed in Supplementary Table 1 .
6
Quantitative Real-Time PCR Analysis of C. albicans
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C. albicans cultures were harvested by centrifugation at 6000 r·min-1 at 4 °C for 5 min. The pellets were flash frozen in liquid nitrogen and stored at −80 °C until RNA preparation. RNA isolation was carried out according to the E.Z.N.A.® Yeast RNA Kit (OMEGA Bio-tek.) instructions. Then 1 µg RNA was subjected to the One Step RNA PCR kit (Takara Inc.) to prepare the cDNA according to the manufacturer’s instructions. The RT-PCR were then proceeded by using the SYBR® PremixEx TaqTM kit (Takara Inc.) with following two-step strategy: (1) 94 °C for 30 s; (2) 40 PCR cycles (94 °C for 30 s, a gene-specific annealing temperature for 30 s). All primer sequences are listed in Table S2 . Real time PCRs of triplicate samples were performed using CFX 96 TouchTM (Bio-Rad, Hercules, CA, USA). The gene expression level relative to the calibrator was expressed as 2−ΔΔCT.
7
Quantitative Gene Expression Analysis
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Total RNA was extracted by Trizol (Invitrogen, Shanghai, China) and cDNA was synthesized by iScriptTM cDNA Synthesis Kit (Bio-Rad, CA, USA) following the instructions of manufacturers. qPCR was conducted using Fast Start Universal SYBR Green Master kit (Roche, Shanghai, China). The total volume of reaction system was 20 μl and prepared as follows: 2× SYBER Green master mix 10 μl, cDNA template 2 μl, forward primer (10 μM) 0.5 μl, reverse primer (10 μM) 0.5 μl, ddH2O 7 μl. Procedures of PCR were as follows: at 95℃ for 30 sec, at 95℃ for 15 sec, at 60℃ for 30 sec, and at 72℃ for 2 min, 40 cycles. PCR was performed in CFX96 TouchTM (#6093, Bio-Rad, CA, USA). Primers were listed in Table 1 .
8
Quantitative Analysis of Plant Gene Expression
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Total leaf RNA was prepared using TRIzol reagent (Invitrogen) and RNase-free DNase (RQ1; Promega) according to the manufacturer's instructions. RNA qualities were assessed by agarose gel electrophoresis and NanoDrop (A260/A280 > 1.8 and A260/A230 > 2.0).32 (link) RT reactions were performed by using an oligo(dT) reverse primer and a reverse transcriptase (Super- scriptII; Invitrogen). The cDNA were assessed by quantitative PCR with 2 sets of housekeeping genes, POLYUBIQUITIN (UBC) and GAPDH.33 (link) Quantitative PCR was performed with the SensiMix SYBR and Fluorescein kit (Bioline) in the CFX96 Touchtm (Bio-Rad) PCR system cycled 40 times by using gene-specific primer sets (Table S2). The annealing temperatures for the primer pairs were 53 °C. To determine the relative abundance of target transcripts, the average threshold cycle (i.e., Ct) was normalized to that of UBC as 2−ΔCt, where −ΔCt = (Ct,gene−Ct,UBC).
9
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated using the TransZol UP reagent (Transgen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized by reverse transcription of purified total RNA using FastQuant RT Kit (Tiangen, Beijing, China). The CFX96 Touch TM (BioRaD, USA) quantitative PCR instrument was used to carry out the quantitative real-time polymerase chain reaction assay. Reactions comprised a 20-μL volume containing 2 μL diluted cDNA, 1 uL forward primer, 1 uL revise primer, and 25 uL 5x UltraSYBR Mixture. Thermal cycler conditions were: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 56°C for 30 s and 72°C for 32 s, and 72°C for 2 min. The specificity of each primer pair was verified by melting curve analysis. The N. benthamiana β-actin gene was used as an internal control. The 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)) was used for quantification, and the variation in expression was estimated from three biological replicates. The primer pairs used for qRT-PCR analysis are listed in Supplemental Table 2
10
Real-Time qPCR and ChIP-qPCR Procedures
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For RT-qPCR, total RNA was isolated using TRIZOL Reagent (Invitrogen, Carlsbad, CA), and cDNA was synthesized using Rever Tra Ace qPCR RT Kit (TOYOBO #FSQ-101, Shanghai, China) for qPCR. For ChIP-qPCR, the ChIP DNA fragments and input genomic DNAs served as temples. qPCR was performed with the SYBR Green PCR master mix (Applied Biosystems, Waltham, MA), and the amplification signals were detected by CFX96 TouchTM (Bio-Rad, Hercules, CA) and analyzed by CFX Manager 3.0 (Bio-Rad). Target gene relative expression level was calculated by 2−ΔCT (ΔCT = CTTarget gene − CTGAPDH) and normalized to the relative expression level detected in control cells. Each sample was tested in triplicate.
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