Samples of the dissected adipose tissue (100 mg) in weight were prepared and processed with RNeasy Mini Kit and RNeasy Lipid Tissue Kit (both QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s protocols. The mRNA was reverse-transcribed, and resultant cDNA was subjected to real-time PCR with gene-specific primers using iQ SYBR Green Supermix and an iCycler real-time PCR machine (both from Bio-Rad, Cressier, Switzerland) according to the manufacturer’s instructions. All primers were purchased from Qiagen. Relative gene expression was analyzed using 2−∆∆Ct method as previously described [43 (link)].
Icycler real time pcr machine
Manufactured by Bio-Rad
Sourced in United States
The iCycler real-time PCR machine is a thermal cycler designed for performing real-time polymerase chain reaction (PCR) experiments. It is capable of quantifying nucleic acid targets in a sample during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Iq sybr green supermix, by Bio-Rad (14 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Trizol, by Thermo Fisher Scientific (9 mentions)
Iscript reverse transcriptase, by Bio-Rad (8 mentions)
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Iq sybr supermix, by Bio-Rad (3 mentions)
Iq sybr green supermix reagent, by Bio-Rad (3 mentions)
Sybr green chemistry, by Bio-Rad (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Sybr green mix ssofast, by Bio-Rad (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Iscript, by Bio-Rad (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Sybr green pcr master mix, by Bio-Rad (2 mentions)
Iscript reverse transcriptase kit, by Bio-Rad (2 mentions)
Miscript sybr green kit, by Qiagen (2 mentions)
Reverse transcript reagents, by Bio-Rad (2 mentions)
Sv total rna isolation system, by Promega (2 mentions)
Itaq sybr green supermix, by Bio-Rad (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
42 protocols using icycler real time pcr machine
1
Quantitative Real-Time PCR Analysis of Adipose Tissue
2
Gene Expression Analysis by qPCR
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Total RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA, USA) and 1
mg of RNA was reverse transcribed using random hexamer primers according to the
manufacturer’s instructions (iScript; Biorad, Hercules, CA, USA). qPCR was
conducted using power SYBR green mix (SsoFast; Biorad, Hercules, CA, USA) with a iCycler
real-time PCR machine (Biorad, Hercules, CA, USA). Primer pairs were selected for their
specificity and efficiency and target gene expression levels were determined by the
comparative cycle threshold method (Ct) or ddCt (dCt of target/dCt of control) method. PCR
controls run in absence of template were consistently negative.
mg of RNA was reverse transcribed using random hexamer primers according to the
manufacturer’s instructions (iScript; Biorad, Hercules, CA, USA). qPCR was
conducted using power SYBR green mix (SsoFast; Biorad, Hercules, CA, USA) with a iCycler
real-time PCR machine (Biorad, Hercules, CA, USA). Primer pairs were selected for their
specificity and efficiency and target gene expression levels were determined by the
comparative cycle threshold method (Ct) or ddCt (dCt of target/dCt of control) method. PCR
controls run in absence of template were consistently negative.
3
Quantitative Real-Time PCR Analysis of Fracture Callus
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Fracture callus tissues were homogenized under liquid nitrogen. Total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). cDNAs were synthesized using an iSCRIPT cDNA Synthesis Kit. Quantitative RT-PCR amplification was performed in an iCycler Real-Time PCR machine using iQ SYBR Green Supermix (all from Bio-Rad). β-Actin was amplified on the same plates and used to normalize the data. Each sample was prepared in triplicate, and each experiment was repeated at least 3 times. The relative abundance of each gene was calculated by subtracting the Ct value of each sample for an individual gene from the corresponding Ct value of β-actin (ΔCt). ΔΔCt was obtained by subtracting the ΔCt from the reference point. These values were then raised to the power 2 (2ΔΔCt) to yield the fold expression value relative to the reference point. Data are presented as the mean ± SD of the triplicates or of 4 wells of cell cultures. The primer sequences and qPCR conditions used are provided in Supplemental Table 2 .
4
Quantifying Mitochondrial DNA in Astrocytes
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Total DNA from primary astrocytes was prepared using Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) and following the manufacturer's instructions. The relative copy numbers of mitochondrial and nuclear DNA were determined by real-time PCR with primers specific to the COX3 (mitochondrial) and 18SrDNA (nuclear) genes, 100 ng DNA, and SYBRGreen PCR master mix (Bio-Rad, Hercules, CA, USA) on an iCycler real-time PCR machine (Bio-Rad).
5
Quantification of Host Gene Expression in Mouse Cecum
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Mouse infections, tissue extraction and quantitative PCR analysis was carried out as previously described [54 (link)]. Briefly, groups of age- and sex-matched 8–12 week-old C57BL/6 mice carrying a wild type allele of Nramp1 (Slc11a1) were administered 100 μl of a 100 μg/ml solution of streptomycin 24 hs prior to bacterial infection. Food was removed 4 hs prior to inoculation, and mice were administered (by stomach gavage) 100 μl of 10% bicarbonate solution (to buffer the stomach pH) followed by the administration of 108 c. f. u. of the indicated bacterial strains in 100 μl PBS. Three days after infection, total RNA from mouse tissues (cecum) were isolated using TRIzol (Invitrogen) reagent according to the manufacture’s protocol and were reversed-transcribed with the iScript reverse transcriptase (BIORAD). Quantitative PCR was performed using iQ SYBR Green Supermix (BIORAD) in an iCycler real time PCR machine (Bio-Rad). All animal experiments were conducted according to protocols approved by Yale University’s Institutional Animal Care and Use Committee.
6
Quantifying Gene Expression in Muscle Atrophy
7
Quantifying Mitochondrial DNA in Astrocytes
8
Testicular Cell Isolation and Analysis
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After dissecting single testicular cells, the cells were resuspended in staining buffer (PBS + 3% FBS) for 20 mins on ice, stained with the primary antibodies, washed with staining buffer, incubated with secondary antibodies for 20 mins on ice, resuspended in staining buffer and sorted by FACS.
For FACS, gating was set based on size (FSC) to remove small debris and doublets. We then used unstained and secondary antibody only (primary omitted) stained as negative controls for gating unstained and false positive stained cells respectively (Figure S3A, middle ).
cDNAs were generated using the Iscript reverse transcriptase (RT) kit, according to the manufacturer’s protocol (Bio-Rad). The RT product and primer pairs (Table S4 ) were mixed with iQ SYBR Green supermix (Bio-Rad) and PCR was performed using an iCycler real-time PCR machine according to the manufacturer’s protocol (Bio-Rad). The production of the amplicon was measured by SYBR green fluorescence and the threshold cycle (Ct) values were calculated. Ct values obtained were normalized to Ct values for the ribosomal protein RPL19 (L19) gene.
For FACS, gating was set based on size (FSC) to remove small debris and doublets. We then used unstained and secondary antibody only (primary omitted) stained as negative controls for gating unstained and false positive stained cells respectively (
cDNAs were generated using the Iscript reverse transcriptase (RT) kit, according to the manufacturer’s protocol (Bio-Rad). The RT product and primer pairs (
9
Quantifying Gene Expression in Brain Regions
10
KRAS Gene Silencing Quantification
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KRAS gene silencing was detected by real-time quantitative PCR assay. Total cellular RNA was isolated using TRIzol (Thermo Fisher Scientific), and 1 μg of total RNA was reverse-transcribed using SuperScript reverse transcriptase (Bio-Rad). PCR was performed on iCYCLER real-time PCR machine (Bio-Rad) using SYBR Green chemistry (Bio-Rad). The genes expression levels were normalized to housekeeping gene GAPDH. The primer sequences are as follows: KRAS forward: 5′-GAC TCT GAA GAT GTA CCT ATG GTC CTA-3′ and reverse: 5′-CAT CAT CAA CAC CCT GTC TTG TC-3′; GAPDH forward: 5′-AAC GGG AAG CTT GTC ATC AAT GGA AA-3′ and reverse: 5′-GCA TCA GCA GAG GGG GCA GAG-3′. Experiments were performed three times.
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