The AB high-capacity cDNA reverse transcription kit (Applied Biosciences by Thermo Fisher Scientific, Vilnius, Lithuania) was used to transcribe RNA to cDNA. The relative expression levels of target genes were analyzed by the Bio-Rad CFX Connect Real-time system with the Bio-Rad CFX Maestro program. Power SYBR Green PCR Master mix (Applied Biosystems, Life Technologies, Woolston Warrington, UK) was used to amplify the genes of 10 ng cDNA per well in combination with 300 nM of respective F/R primer set (Table 2 ). The PCR cycle involved an initial heating at 50 °C for 2 min followed by an activation step at 95 °C for 10 min and then 40 cycles of amplification (95 °C for 15 s and 60 °C for 1 min). The dissociation curve was determined by initial heating at 95 °C for 15 s, followed by 10 s at 60 °C, with 0.5 temperature increments until 95 °C was reached. gyrA served as the housekeeping gene [44 (link)] for calculating the relative changes in target gene expression by the 2−ΔΔCt method. The fold change of each gene for each treated sample was analyzed against each control sample, and the averages of the obtained values are presented. Gene expression is expressed in relative values, with the expression level of the control sample set to 1 for each gene. The assays were performed in triplicate and repeated three times [13 (link)].
Cfx maestro program
Manufactured by Bio-Rad
Sourced in United States
The CFX Maestro program is a software application developed by Bio-Rad for use with their CFX real-time PCR detection systems. It provides a user interface for instrument control, data acquisition, and analysis of real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Cfx connect real time system, by Bio-Rad (6 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Genelute bacterial genomic dna kit, by Merck Group (2 mentions)
Qscript cdna synthesis kit, by Quantabio (1 mentions)
Cfx opus 96, by Bio-Rad (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Iscript kit, by Bio-Rad (1 mentions)
Ssoadvanced sybr green, by Bio-Rad (1 mentions)
C1000 touch thermal cycler instrument, by Bio-Rad (1 mentions)
Nanodrop device, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Superreal premix color, by Tiangen Biotech (1 mentions)
Maxima sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions)
Cfx384 real time pcr system, by Bio-Rad (1 mentions)
48 well tissue culture plate, by Corning (1 mentions)
11 protocols using cfx maestro program
1
Quantitative Real-Time PCR Analysis
2
Quantitative gene expression analysis protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RNA was converted to cDNA using the cDNA qScript cDNA synthesis kit (QuantaBio, Beverly, MA, USA). The relative expression levels of target genes were analyzed by Bio-Rad CFX Connect Real-time system with the Bio-Rad CFX Maestro program. Power SYBR Green PCR Master mix (Applied Biosystems, Waltham, MA, USA) was used to amplify the genes of 10 ng cDNA per well in combination with 300 nM of respective F/R primer set (Table 1 ). The PCR cycle involved initial heating at 50 °C for 2 min and an activation step at 95 °C for 10 min, followed by 40 cycles of amplification (95 °C for 15 s and 60 °C for 1 min), and the dissociation curve was determined by initial heating at 95 °C for 15 s, followed by 10 s at 60 °C, and 0.5 temperature increments until 95 °C was reached. The 16S rRNA expression was used for normalization and to calculate the relative changes in target gene expression using the 2−ΔΔCt method. Control reactions were performed with RNA that had not been reverse-transcribed to ensure that no genomic DNA was amplified during the PCR process. Gene expression was expressed in relative values, setting the expression level of the untreated and ethanol-treated control to 1 for each gene. The assays were performed in triplicates and repeated three times [40 (link)].
3
Quantitative Analysis of snRNA Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
snRNA variants were quantitatively measured following the procedure mentioned above. The following amounts of RNA per RT-qPCR reaction were used: total cell RNA 5.0 ng, Sm RIP 1.0 ng RNA, cytoplasmic RNA 5.0 ng, gradient fractionation of snRNPs and spliceosomes 0.5–1.0 ng RNA. Data preprocessing was carried out using Bio-Rad's CFX Maestro program. Unreliable RT-qPCR values (>35) were removed from downstream analysis. Subsequent data analysis was carried out following standard ΔCt or ΔΔCt approaches as indicated in figure legends or specified below.
4
Quantitative RT-PCR Protocol for Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The purified RNA was reverse transcribed into cDNA using the cDNA qScript cDNA synthesis kit (QuantaBio). The relative expression levels of target genes were analyzed by Bio-Rad CFX Connect Real-time system and the Bio-Rad CFX Maestro program. Power Sybr Green PCR Master mix (Applied Biosystems) was used to amplify the genes of 10 ng cDNA per well in combination with 300 nM of respective F/R primer set (Table 2 ). For each set of primers, a standard amplification curve (critical threshold cycle vs. exponential of concentration) was plotted, and only those with a slope around –3.2 were used for analysis. The PCR cycle involved initial heating at 50°C for 2 min, activation step at 95°C for 10 min, followed by 40 cycles of amplification (95°C for 15 s, 60°C for 1 min), and the dissociation curve was determined by initial heating at 95°C for 15 s, followed by 10 s at 60°C, and 0.5 temperature increment until reaching 95°C. The expression of 16S rRNA was used for normalization and to calculate the relative changes in target gene expression using the 2−ΔΔCt method. Gene expression was expressed in relative values, setting the expression level of the untreated control to 1 for each gene (Assaf et al., 2014 ; Feldman et al., 2014 (link)).
5
Quantitative Real-Time PCR Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative real-time PCR was performed as described by Aqawi et al. [35 (link)]. Each reaction was prepared by mixing 10 ng cDNA with 300 nM of respective forward and reverse primers (Supplementary Table S1 ) and Power Sybr Green PCR Master mix (Applied Biosystems, Warrington UK). Triplicates were done for each gene of each sample. The amplification was done in a Bio-Rad CFX Connect Real-time system using the Bio-Rad CFX Maestro program with 40 cycles of 15 sec at 95 °C and 1 min at 60 °C. The 16S rRNA was used as a housekeeping gene and the changes in gene expression were calculated using the 2−ΔΔCt method. The control was set to 1 for each gene, and gene expression is presented as relative values.
6
Quantifying Vibrio harveyi Biofilm Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA extraction and quantification were performed as previously described (Periasamy and Kolenbrander, 2009 (link); Assaf et al., 2014 ). Briefly, biofilm was allowed to form in the absence or presence of different concentrations of CBG and ethanol in polystyrene 48-well tissue culture plates (Nunc). At the end of incubation, the growth medium was removed, and the formed biofilms were washed twice with PBS. Then, 200 μl of NaOH (0.04 M) was added to each well and the plates were incubated in a hot water bath for 1 h at 60°C, followed by neutralization with 18.5 μl Tris:HCl pH 7.0. The amount of DNA in each sample was quantified by qPCR with specific primers for V. harveyi 16S rRNA (Table 2 ) using a Bio-Rad CFX Connect Real-time system and the Bio-Rad CFX Maestro program. The amount of DNA in each sample was calculated according to the standard curve obtained using known DNA concentrations of purified V. harveyi DNA. Purified DNA was extracted from an overnight culture of V. harveyi BB120 using GenElute Bacterial Genomic DNA kit (Sigma Aldrich, St. Louis, MO, United States) as per the manufacturer’s instructions.
7
Quantifying Gene Expression from Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each RNA sample was isolated using RNeasy Micro Kit (QIAGEN, #74004) from 30 wild-type (WT) embryos. cDNA was synthesized with the iScript kit (Bio-Rad, #1708841) using 1mg of total RNA. qRT-PCR reactions were set up using SsoAdvanced SYBR green (Bio-Rad, #1725270) on CFX Opus 96 (Bio-Rad) and the gene expression was analyzed applying CFX Maestro program (Bio-Rad). Primers used are listed in Supplementary materials Table S1 .
8
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative RT-PCR (qPCR) analysis was used for further validation of the expression of selected genes. RNA concentration and purity was measured using a NanoDrop device (ND-1000; Nano Drop Technologies, Wilmington, Delaware). cDNA was prepared using 1 µg of total RNA and the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) following the manufacturer’s instructions. Validated primers for selected genes were purchased from BioRad (Hercules, CA) and qPCR analysis was performed on a C1000 Touch Thermal Cycler instrument (Bio-Rad, Hercules, CA) using the iTaq Universal SYBR Green Supermix (Bio-Rad). Each sample was run in duplicates and qPCR data analysis was performed using the Bio-Rad CFX Maestro program. Gene expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the levels of gene expression were quantified using the 2−ΔCT method
9
Dissecting DSF's Influence on Ovarian Granulosa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To explore the possible mechanism through which DSF regulates the reproductive capacity of female mice, ovarian granulosa cells were cultured in cell culture medium containing different concentrations of DSF (0, 0.25 μM, 0.5 μM, 1 μM or 10 μM). After culturing for 24 h, a total RNA extraction kit was used to extract RNA from granular cells in different concentration groups, and the concentration and purity of RNA were determined to be within the available range. The number of granular cells in each sample was about 2 × 106. A reverse transcription kit was used to reverse-transcribe RNA into cDNA for real-time fluorescence quantitative assay. The relative mRNA expression of each gene was detected with qRT-PCR. The primer sequences of genes were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome ) (accessed on 17 October 2020), with β-actin used as a housekeeping gene. The mRNA expression of related genes was detected using SuperReal PreMix Color (Tiangen Biotech, Beijing, China) and qRT-PCR conditions of 95 °C for 15 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 32 s). The detection results were analyzed with the Bio-Rad CFX Maestro program.
10
Quantification of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from LV tissue and real‐time PCR was performed using the CFX384™ Real Time PCR System (BioRad, USA) and Maxima SYBR Green qPCR Kit (Thermo Scientific, Germany) as recently described.25 Specific primer sequences are listed in TableS2 . The expression of specific genes was normalized to its expression in lean. The CFX Maestro program (BioRad, USA) was used to calculate relative gene expression.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!