Primers were designed from previously reported cDNAs or genes from RNA-Seq data of T. salsuginea. We designed gene-specific primers using PRIMER 5.0 software (Table S6 ). RT-PCR was used to confirm primers quality and PCR amplification efficiency. We used 1 µg total RNA to synthesize first-strand cDNA using the cDNA Synthesis Kit (TransGen Biotech, Beijing, China). Real-time PCR was performed using TransStart® Top Green qPCR SuperMix (TransGen Biotech, Beijing, China) on the Bio-rad system (Hercules, CA, USA). The amplification procedures were as follows: 95 °C for 1 min, followed by 40 cycles at 95 °C for 30 s, 58 °C for 30 s. All samples were assayed in triplicate wells. Data acquisition and analysis were performed for each run using ICYCLERIQ software (version 3.0a Bio-Rad, Hercules, CA, USA). To normalize the differences in the total RNA amount, UBQ5 was used as an internal control [69 (link)]. The 2−△△Ct method was used to calculate the relative expression change [70 (link)].
Icycler iq software
Manufactured by Bio-Rad
Sourced in United States
The iCycler iQ software is a real-time PCR detection system designed for quantitative gene expression analysis. It provides advanced thermal cycling and data analysis capabilities for a wide range of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Icycler thermocycler, by Bio-Rad (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Cdna synthesis kit, by Transgene (1 mentions)
Transstart top green qpcr supermix, by Transgene (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Cfx connect thermal cycler, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnaprotect bacteria reagent, by Qiagen (1 mentions)
Iscript one step rt pcr kit with sybr green, by Bio-Rad (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Pasw statistics 18, by IBM (1 mentions)
Rnalater, by Qiagen (1 mentions)
Icycler, by Bio-Rad (1 mentions)
Rneasy mini, by Qiagen (1 mentions)
Multiscribe rt, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix kit, by Bio-Rad (1 mentions)
Icycler iq multicolor real time pcr detection system, by Bio-Rad (1 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
Biorad iq cycler, by Bio-Rad (1 mentions)
Iq cycler, by Bio-Rad (1 mentions)
14 protocols using icycler iq software
1
Real-time qPCR Analysis of T. salsuginea
2
SYBR Green qPCR for Gene Expression
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The PCR reactions were carried out in a final volume of 20 μL, containing 100 nM of each primer and 2 μL of cDNA (synthetized in the previous step) in iQ™ SYBR® Green Supermix (Bio-Rad, Madrid, Spain). Following an initial 3 min denaturation/activation step at 95 °C, the mixture was subjected to 45 cycles of amplification (denaturation for 15 s at 95 °C; annealing and extension for 15 s at 58 °C) in a CFX Connect™ Thermal Cycler (Bio-Rad, Madrid, Spain). The generation of PCR products was monitored after each extension step at 58 °C by measuring the fluorescence of double-stranded DNA binding SYBR Green dye. In order to determine the melting temperatures of the amplified products after SYBR Green qPCR, the temperature was raised from 55 °C to 95 °C and the fluorescence was detected for 10 s after each 0.2 °C. From each reaction, the threshold cycle value (Ct) was established as the cycle number at which fluorescence was detectable over the threshold value calculated by the iCycler iQ™ software (Bio-Rad, Madrid, Spain) for cycles 2–10.
3
Quantitative RT-PCR for Bacterial RNA Profiling
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qRT-PCR was carried out as previously described (33 (link)). Bacterial cultures grown to mid-exponential phase in LB at 37°C were added directly to RNAprotect Bacteria Reagent (Qiagen) according to the instructions of the manufacturer to stabilize RNA. Total RNA was purified with an RNeasy minikit (Qiagen), and DNA was removed with a Turbo DNA-free kit (Ambion). An IScript One-Step RT-PCR kit with SYBR green (Bio-Rad) was used to measure RNA or DNA control levels according to the manufacturer’s instructions. Reaction mixtures (20 µl) contained 100 ng of RNA (or an appropriate concentration of DNA standard), 1× SYBR green RT-PCR reaction mix, and a 300 nM concentration of each primer. The thermal cycling was done with an iCycler thermocycler (Bio-Rad) with 10 min reverse transcription at 50°C for 2 min at 95°C and then 45 cycles of PCR at 95°C for denaturation for 10 s and at 60°C for annealing, extension, and detection for 20 s. Melting curves were used to verify the specificity of the PCR product after the qRT-PCR reaction. ICycler iQ software (Bio-Rad) was used to determine RNA abundance relative to a standard curve of PCR products and 16S rRNA levels.
4
Somatic Embryo Development Analysis
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All experiments were repeated at least twice independently to ensure the reproducibility of the results. Data on the percentages of grapevine somatic embryos in the different developmental stages were statistically analyzed using a Mann-Whitney U test. Data about the relative water content were statistically analyzed using a Kruskal-Wallis test followed by a Bonferroni post hoc test. Data on ABA and ABA-GE contents in the somatic embryo aggregates were statistically analyzed using one-way ANOVA with the Student-Newman-Keuls post hoc test. Statistical tests (P < 0.05) were performed using PASW Statistics 18 software (IBM, New Orchard Road, New York, USA).
Data from the qPCR were analyzed using iCycler iQ™ software (Real-Time Detection System Software (Bio-Rad, Windows ver. 3.0). The raw fluorescence data were analyzed using LinRegPCR software [42 (link)] to obtain the mean PCR efficiency for each primer pair. Relative gene expression was determined and statistically analyzed (P < 0.05) using the REST-2009© (Relative Expression Software Tool, ver. 2009, [71 (link)]) with PCR efficiency correction and normalization by two reference genes as validated for this plant material in the present work, and compared with the 2-ΔΔCq method.
Data from the qPCR were analyzed using iCycler iQ™ software (Real-Time Detection System Software (Bio-Rad, Windows ver. 3.0). The raw fluorescence data were analyzed using LinRegPCR software [42 (link)] to obtain the mean PCR efficiency for each primer pair. Relative gene expression was determined and statistically analyzed (P < 0.05) using the REST-2009© (Relative Expression Software Tool, ver. 2009, [71 (link)]) with PCR efficiency correction and normalization by two reference genes as validated for this plant material in the present work, and compared with the 2-ΔΔCq method.
5
LCMV RNA Quantification from Spleen Samples
6
Quantitative RT-PCR Analysis of Rat MMPs
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The forward and reverse primer sequences of rat MMPs used in this study has been reported earlier by our group41 (link). Reaction set-up for each cDNA sample was assembled using the IQ™ SYBR® Green Supermix kit (Bio-Rad Laboratories, CA) as per the manufacturer's instructions. Samples were subjected to forty cycles at 95°C for fifteen seconds and 60°C for one min in iCycler IQ (Multi Color Real-Time PCR Detection System, Bio-Rad Laboratories, CA). Data were collected and recorded using the iCycler IQ software (Bio-Rad Laboratories, CA) and expressed as a function of the threshold cycle (Ct), which represents the number of cycles at which the fluorescent intensity of the Sybr Green dye is significantly above than that of the background fluorescence. The housekeeping gene, β-actin was used for normalization of MMPs expression. Average Ct values were normalized with average Ct values of β-actin. After normalization of the Ct values, fold differences were calculated by using the formula 2^-(ΔCt of Test)/2^-(ΔCt of controls).
7
APOE Genotyping by TaqMan-PCR
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Genomic DNA was extracted from peripheral blood leukocytes. APOE rs429358 and rs7412 genotypes were determined by TaqMan polymerase chain reaction (TaqMan-PCR) using an iCycler iQ real-time PCR detection system (iQ5; Bio-Rad, Hercules, CA, USA). Each PCR was performed in 25 μL total volumes consisting of 1 × TaqMAN® Universal Master Mix II (cat#4440041; Applied Biosystems [ABI], Foster City, CA, USA), 0.2 μM each probe and each primer (cat# 4351379; ABI), and 50–100 ng genomic DNA. PCR programs had an initial denaturation of 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Data analysis for allele discrimination was performed using iCycler iQ software (Bio-Rad). Controls were included in each run for quality control, and repeated genotyping of random 5% subsets yielded 100% identical genotypes. Additionally, sequencing analyses performed using the ABI 3730XL DNA Sequencing System with universal primers (5′-GGCGC GGACA TGGAG GAC-3′ and 5′-GCCCC GGCCT GGTAC ACT-3′) for both polymorphisms yielded 100% identical genotypes.
8
Quantifying Leiomyoma Gene Expression
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Quantitative real-time PCR was performed to examine Ki67, PR-A and PR-B, ERK, Akt, PTEN, PPARγ, mRNAs expression in leiomyoma tissue explants and primary culture cells after incubation with progesterone. Total RNA was extracted from samples using the standard acid guanidinium thiocyanate-phenol-chloroform method. The amount of RNA in each sample was estimated with a Nano Drop 2000C (Thermo Scientific, USA) spectrophotometer. Five micrograms of RNA was reverse-transcribed into cDNA using a commercial kit (Fractal Bio, St. Petersburg, Russia), according to the manufacturer’s protocol. Real-time PCR was performed with iCycler IQ software (BIO-RAD, CA, USA). Commercial kits for quantitative estimation of specific gene mRNAs expression (Fractal Bio, St. Petersburg, Russia) were used. Quantification was performed for the expression of β-actin, a compound expressed consistently in uterine leiomyoma tissue and leiomyoma cells used as housekeeping gene. A series of control DNA dilutions were used to generate standard curves and the number of copies of cDNA of specific genes was defined and normalized to β-actin expression. Results for mRNAs expression of the studied genes are presented as a normalized number of copies x103 per µl.
9
Real-Time SYBR Green PCR Analysis
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Real-Time PCR was performed in a Bio-Rad iQ-cycler (Bio-Rad) equipped with skirted Micro seal 96-Well PCR-plates covered with Microseal ‘B’ adhesive foils (Bio-Rad). Reaction volume (20 μl, containing 30 ng cDNA) consists of 4 μl diluted cDNA-solution (7.5 ng/μl), 2 μl 0.1 μM gene-specific forward and reverse primer solution (2 pMol each; Biomers, Ulm, Germany), 4 μl DEPC-treated water and 10 μl RT2 (link) Real-Time SYBR-Green/Fluorescein-PCR-Master-Mix (SA Bioscience, Hilden, Germany/Qiagen). According to manufacturer recommendations, a cycling program with an initial activation step (94 °C, 3 min) followed by 45 cycles of denaturation (94 °C, 30 s), annealing (60 °C, 30 s), elongation (72 °C, 30 s) and a final elongation (72 °C, 30 s) is executed. Fluorescence acquisitions in the SYBR green and ROX (internal reference dye) channels were performed at the end of the annealing step. A melting protocol ranged from 94 to 59 °C following a stepwise increment of 0.5 °C held for 3 s. Each sample as well as a negative template control (NTC) was amplified in triplet for each of the primer pairs assayed. Raw data (ct-values) were extracted using iCycler iQ-software (version 3.1, Bio-Rad) running on the Bio-Rad iQ-cycler.
10
Quantitative Expression Analysis of Dental MSCs
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Total RNA was extracted from 3 dental MSCs-loaded scaffolds at each time point with NucleoSpin kit (NucleoSpin RNA, Macherey-Nagel, Germany), as recommended by the manufacturer. Subsequently, cDNA synthesis was obtained with the iScriptTM cDNA Synthesis Kit (BioRad, United States) as recommended by the manufacturer. After cDNA synthesis reaction, quantitative real-Time PCR was carried out in mixture containing 1 μL of cDNA, 10 μM of each forward and reverse primers (Supplementary Table 2 ) and 10 μL of iTaqTM Universal SYBR® Green Supermix (BioRad, United States). qPCR experiments were run using an iQ5 (BioRad, United States) and analyzed with the iCycler IQ software (BioRad, United States). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous assay control. Relative quantification of gene amplification by qPCR was performed using the cycle threshold (Ct) values and relative expression levels were calculated using the 2∧(−ΔΔCT) method. For each PCR, samples were analyzed in duplicate and three independent experiments were performed.
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