Total RNA was extracted from root tissues using the Trizol reagent (Invitrogen, Waltham, MA, United States) according to the manufacturer’s instructions, and the purified RNA was treated with RNase-free DNase I (Promega, Madison, WI, United States) to remove any DNA contamination. The expression patterns of selected genes were determined using quantitative real-time reverse transcription PCR (qRT-PCR) as described previously (Cai et al., 2015 (link)) using a qPCR CFX 96 Thermocycler (Bio-Rad, Hercules, CA, United States). The EF1α gene was used as the reference gene to normalize the relative quantification. The expression ratio and fold change (FC) were calculated using the 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)). All reactions were performed in triplicate. The gene-specific primers are shown in Supplementary Table 3 .
Qpcr cfx 96 thermocycler
Manufactured by Bio-Rad
Sourced in United States
The QPCR CFX 96 Thermocycler is a real-time PCR (qPCR) instrument designed for quantitative gene expression analysis. It features a 96-well format and can perform precise temperature cycling for DNA amplification and detection.
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5 protocols using qpcr cfx 96 thermocycler
1
Quantitative analysis of gene expression
2
Thermal Stability Assay for (Ec)PycC
3
Thermal Stability Profiling of TREX1 Proteins
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10 μM TREX1 or TREX1 chimeras was supplemented with 3× SYPRO Orange Dye (Life Technologies) in a 20 μL reaction buffer containing 20 mM Tris-HCl pH 7.5, 75 mM KCl, and 1 mM TCEP. Reactions were incubated with an increasing temperature from 20 to 95 °C over ~2 h using a qPCR CFX96 thermocycler (Bio-Rad). Fluorescence in the HEX channel was measured every 0.5 °C and melting temperature (Tm) was defined as the temperature at which the half of the maximum fluorescence change occurs. For TREX1-DNA binding analysis, 10 μM TREX1 or TREX1 mutants was incubated with a 30-bp dsDNA (10 or 20 μM; sequence: 5′-GCTCGAGTCATGACGCGTCATGACTCGAGC-3′) at 4 °C for 30 min, and the reactions were subjected for thermal denaturation analysis as previously describe44 .
4
Targeted Silencing of BRCA1 in U2OS Cells
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Small interference RNAs (siRNAs) targeting human BRCA1 and a ‘Scramble’ siRNA as control were purchased from RiboBio Co. At 3 days after transfection of 1.0 × 105 U2OS cells with 20 pmol siRNA together with the Cas9/nCas9-sgRNA expression plasmids, RNAs were isolated and reverse-transcribed to complementary DNA using the HiScript II Q RT SuperMix for qPCR (Vazyme). Quantitative reverse transcriptase PCR (qRT-PCR) was performed for siRNA-mediated BRCA1 depletion on qPCR CFX 96 Thermocycler (Bio-Rad) using gene-specific primers (Supplementary Table 2 ).
5
Diagnostic Potential of miR-21-5p in Prostate Cancer
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Quantitative PCR was conducted using an ExiLent SYBR Green Master Mixkit (Exiqon, Denmark), primersset (forward and reverse), and diluted cDNA. The qPCR analysis of gene expression was performed using the qPCRCFX96 thermocycler (Bio-Rad). All of the procedures followed the manufacturer's recommendations, and statistical analyses were performed using the SPSS Version 23 and Graph Pad Prism 7. In this study, statistical significance was setata p-value <0.05 (Table 1 and2 1).
The expressions of each biomarker were analyzed using Mann Whitney Test. We found a significant difference between miR-21-5p expression in the BPH group and PCa, both metastatic and non-metastatic, withp-values of 0.017 and 0.004, respectively. Never theless, expression of miR-21-5p between metastatic and nonmetastasic PCa showed in significant results. BPH group showed the highest expression of PDCD-4 mRNA. Significant difference was found between BPH group and PCa group, both metastatic and nonmetastatic.
In this study, miR-21-5p showed potential diagnostic value todetect PCa. The expression of miR-21-5p, using 1.34 as cut-off point had 83% sensitivity and 44.4% specificity.
The expressions of each biomarker were analyzed using Mann Whitney Test. We found a significant difference between miR-21-5p expression in the BPH group and PCa, both metastatic and non-metastatic, withp-values of 0.017 and 0.004, respectively. Never theless, expression of miR-21-5p between metastatic and nonmetastasic PCa showed in significant results. BPH group showed the highest expression of PDCD-4 mRNA. Significant difference was found between BPH group and PCa group, both metastatic and nonmetastatic.
In this study, miR-21-5p showed potential diagnostic value todetect PCa. The expression of miR-21-5p, using 1.34 as cut-off point had 83% sensitivity and 44.4% specificity.
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