According to the standard protocol, the total cell RNA solution was extracted with Trizol (Invitrogen, Carlsbad, United States). RNA concentration was validated by Nanodrop (Invitrogen, United States), followed by in vitro cDNA synthesis with PrimeScriptTM RT Master Mix kits (Takara, Japan). Then SYBR® Select Master Mix kit (designed for total RNA) was used for RT-qPCR analysis on the Bio‐Rad CFX96 qPCR instrument (Bio‐Rad, Hercules, CA). β-Actin was selected as a reference gene for the coding mRNA test, while U6 was used as a reference gene for the microRNA test. The primers were: PTEN-forward:5′-CAA GAT GAT GTT TGA AAC TAT TCC AAT G-3′; PTEN-Reverse: 5′-CCT TTA GCT GGC AGA CCA CAA-3′; miR-21-5′-GCC CGC TAG CTT ATC AGA CTG ATG-3′; β-actin-F-5′-CTG AAC CCT AAG GCC AAC CG-3′; β-actin-R-5′-GTC ACG CAC GAT TTC CCT CTC-3′; U6-F-5′-GCT TCG GCA GCA CAT ATA CTA AAA T-3′; U6-R-5′-CGC TTC ACG AAT TTG CGT GTC AT-3′.
Cfx96 qpcr instrument
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Germany, China
The CFX96 qPCR instrument is a real-time PCR detection system designed for quantitative gene expression analysis. The instrument utilizes advanced optics and thermal cycling technology to accurately measure the amplification of DNA sequences in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Itaq universal sybr green supermix, by Bio-Rad (8 mentions)
Nanodrop, by Thermo Fisher Scientific (7 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Iscript rt supermix, by Bio-Rad (4 mentions)
Ssoadv univer sybr grn master kit, by Bio-Rad (4 mentions)
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
Quick rna miniprep kit, by Zymo Research (4 mentions)
Sybr select master mix kit, by Bio-Rad (3 mentions)
Ssofast evagreen supermix, by Bio-Rad (3 mentions)
Ds 11 spectrophotometer, by DeNovix (3 mentions)
Genomic mini ax body fluids kit, by A&A Biotechnology (2 mentions)
K3edta tube, by BD (2 mentions)
Primescript rt master mix kits, by Bio-Rad (2 mentions)
Mirna assay kit, by Bio-Rad (2 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Rnase free dnase 1, by New England Biolabs (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
66 protocols using cfx96 qpcr instrument
1
Quantification of PTEN and miR-21 Expression
2
Quantification of Cell-free DNA and HPV16
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For measurement of the total circulating cell-free DNA in blood (cfDNA), the amplification of TERT (human telomerase reverse transcriptase) was used as described previously [8 (link)]. Shortly, each measurement consisted of a standard curve, negative control and a sample. For the construction of the standard curve, we used a control genomic DNA. The concentration of the total cfHPV DNA did not influence the cfHPV DNA detection as we presented before [8 (link)]. For HPV16 detection, independent reaction was performed using primers and probe set for HPV16 genome. Each measurement consisted of a standard curve, negative control and a sample. A standard curve using tenfold DNA dilutions of plasmid construct containing HPV16 genome was plotted. The obtained copies of cfHPV DNA were calculated according to the amount of plasma that was taken for DNA extraction (copies/ml). PCR reactions were performed using the Bio-Rad CFX96 qPCR instrument (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom). If HPV16 was found, it’s presence would be confirmed with a second independent DNA isolation.
3
Quantification of TLR2 and TLR4 Expression
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Total mucosal RNA was extracted from the colonic biopsies using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan), according to the manufacturer’s instructions. After reverse transcription with PrimeScript Reverse Transcriptase Mix (TaKaRa), the expression of TLR2 and TLR4 genes was determined by quantitative real-time polymerase chain reaction (qPCR) and SYBR Green technology on a Bio-Rad CFX96 Q-PCR instrument (Bio-Rad, United States) in duplicate. The specific primers for TLR2 were: 5’-TGATGCTGCCATTCTCATTC-3’ (forward) and 5’-CGCAGCTCTCAGATTTACCC-3’ (reverse); and for TLR4: 5’-CAGGGCTTTTCTGAGTCGTC-3’ (forward) and 5’-TGAGCAGTCGTGCTGGTATC-3’ (reverse). Each amplification reaction was run in duplicate in a final volume of 20 μL. All qPCR amplifications were optimized and performed in 0.2 mL 96-well plates with the following cycling program: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Negative controls lacking the template DNA were included in triplicate. The relative amount of each mRNA was normalized to GAPDH (forward: 5’-CCATCAATGACCCCTTCATTG-3’, reverse: 5’-CTTGACGGTGCCATGGAATT-3’) and data were analysed using the 2-ΔΔCT method.
4
Watermelon Fruit Tissue Total RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from watermelon fruit tissue in three biological replicates using RNeasy® Plant Mini Kit (QIAGEN Sciences, Germantown, MD, USA) as per the manufacturer’s protocol. The first strand cDNAs were synthesized with 1 μg of total RNA in 20 μL of the reaction mixture using iScript RT Supermix (Bio-Rad Laboratories, Inc, Hercules, CA, USA) as per the manufacturer’s instructions. Gene expression analysis via reverse transcription-qPCR was performed using the BioRad CFX96 qPCR instrument and the SsoAdv Univer SYBR GRN Master Kit (Bio-Rad Laboratories, Inc, Hercules, CA, USA). The watermelon β-actin was used as an internal control, and the relative expression levels (Cq values) for each gene were normalized to β-actin transcription. The relative expression levels were calculated using the ΔΔCq (quantitative cycle) method provided with the Bio-Rad CFX software. The primers used for qRT-PCR analysis are listed in Table S1 .
5
Quantitative HPV16 DNA Detection
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Peripheral blood (12 mL) was collected into K3EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Plasma was separated within an hour by double centrifugation at 300× g and 1000× g, both at 4 °C for 10 min. DNA was extracted (according to the manufacturer’s instructions) from 1 mL of plasma by the Genomic Mini AX Body Fluids kit (A&A Biotechnology, Gdynia, Poland). Each measurement consisted of a standard curve of three dilutions of plasmid construct containing HPV16 genome, negative control and a samples. For HPV16 detection, reaction was performed using primers and probe set for the HPV16 genome. PCR reactions were performed using the Bio-Rad CFX96 qPCR instrument (Bio-Rad Laboratories, Hemel Hempstead, UK). If HPV16 was found, its presence would be confirmed with a second independent DNA isolation.
6
Comprehensive RNA Extraction and Quantification
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Trizol purchased from Invitrogen (Calsbad, USA) was used to extract total cell RNA as standard protocol. RNA concentration was tested by Nanodrop (Invitrogen, USA). PrimeScriptTM RT Master Mix kits were used to synthesis cDNA; and Taqman MirNA assay kit (designed for microRNA) and SYBR® Select Master Mix kit (designed for total RNA) were used for RT-qPCR analysis on Bio-Rad CFX96 qPCR instrument (Bio-Rad,Hercules, CA). All these kits were purchased from Takara (Japan). β-actin and U6 were used as reference genes.
7
Plasma-based HPV16 Detection
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Peripheral blood (12 mL) was collected in K3EDTA tubes (Becton–Dickinson, Franklin Lakes, NJ, USA). Plasma was separated within an hour by double centrifugation at 300× g and 1000× g, both at 4 °C for 10 min. DNA was extracted (according to the manufacturer’s instructions) from 1 mL of plasma using the Genomic Mini AX Body Fluids Kit (A&A Biotechnology, Gdynia, Poland). Each measurement consisted of a standard curve of three dilutions of a plasmid construct containing the HPV16 genome, a negative control, and a sample. For HPV16 detection, a reaction was performed using primers and a probe set for the HPV16 genome. PCR reactions were performed using the Bio-Rad CFX96 qPCR instrument (Bio-Rad Laboratories, Hemel Hempstead, UK). If HPV16 was found, its presence was confirmed with a second independent DNA isolation.
8
Optimized qPCR Amplification Protocol
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After confirming primer specificity by amplicon sequencing, qPCR reactions were optimized using a Bio‐Rad CFX96 qPCR Instrument (Bio‐Rad Laboratories). The primer performance was initially evaluated using a gradient of melting temperatures (Tm) in a reaction volume of 10 µl containing 200 nM of each primer, 2 µl of DNA (80 ng DNA/2 µl) for the samples, or 2 µl of serial dilutions (corresponding to 102–107 template copies in the reaction) for the standard curve, 5 µl of 2× Forget‐Me‐Not™ EvaGreen® qPCR Master Mix (Biotium) and 2.4 µl of sterile nuclease‐free water. Thermocycling conditions were 95°C for 2 min, followed by 40 cycles of 95°C for 5 s, the optimized melting temperature for each primer pair for 10 s (between 58 and 61°C) and 72°C for 10 s. A final melting curve was generated by increasing the temperature up to 95°C in increments of 0.5°C every 10 s to confirm single reaction products were obtained. Control reactions included substitution of genomic DNA by water to confirm the absence of contamination.
9
Relative Quantification of Gene Expression
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Cell Total RNA isolation kit (FOERGENE, Chengdu, China) was used to extract total RNA, and then reverse-transcribed into cDNA by Reverse transcription kit (ABclonal, Wuhan, China). Genious 2x SYBR Green Fast qPCR mix (ABclonal, Wuhan, China) was used to perform qPCR and detected by Bio-Rad CFX96 qPCR instrument (Bio-Rad, Hercules, CA, USA). In a previous study, we found that PFDN5 and TBP genes were the most stable reference genes for BAT to WAT transformation through RNA-seq [35 (link)]. Thus, TBP and PFDN5 genes were applied as reference genes. Primers used in this study were shown in Table S1 . The relative expression levels of the target genes were normalized relative to the expression of PFDN5 and TBP using the 2−ΔΔCT method. Six biological replicates and three technical replicates in a biological sample were used for qPCR.
10
Quantitative PCR for HPV16 Copy Number
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To evaluate HPV16 copy number per tumor genome, genomic DNA extracted from fresh frozen biopsy or fine needle aspiration specimens was analyzed with quantitative PCR using the HPV Genotypes 14 Real‐TM Quant kit (Sacace Biotechnologies, Como, Italy) and Bio‐Rad CFX96 qPCR instrument (Bio‐Rad) according to the manufacturer's instructions. HPV16 copy number per tumor genome was calculated with reference to the β‐globin copy number.
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