Copy numbers of genes encoding the denitrification associated enzymes nitrite reductase (nirK/nirS) and nitrous oxide reductase (nosZ), nitrogen fixation associated dinitrogenase (nifH), nitrification associated archaeal and bacterial ammonia monooxygenase (amoA), nitrite reductase associated with the dissimilatory reduction of nitrate to ammonia (nrfA) as well as archaeal and bacterial 16S rRNA were quantified by qPCR. Primers and PCR conditions used are given in Table S1 . A typical reaction mixture contained 12.5 μL of SybrGreen Jump-Start ReadyMix (Sigma-Aldrich, Taufkirchen, Germany), 0.5 μM of each primer, 3–4.0 mM MgCl2, 2 μL of soil DNA except for amplification of nosZ, for which 3 μL of DNA were used. For the amplification of functional marker genes involved in nitrogen cycling 200 ng BSA mL−1 were added. All assays were performed in an iCycler (Applied Biosystems, Darmstadt, Germany). Standard curves were obtained using serial 10-fold dilutions of a known amount of plasmid DNA (108 to 101 gene copies) containing the respective gene fragment. Negative controls were always run with water instead of template DNA. PCR reactions were done with 1:50 and 1:100 diluted DNA extracts. Efficiencies for all assays were between 80 and 97% with r2-values between 0.971 and 0.996.
Icycler
Manufactured by Thermo Fisher Scientific
Sourced in United States
The iCycler is a real-time PCR detection system designed for DNA amplification and detection. It features a thermal cycler and a fluorescence detection system for performing quantitative PCR (qPCR) analysis. The iCycler can be used to quantify nucleic acid samples and monitor the progress of PCR reactions in real-time.
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Lab products found in correlation
Pcr sprint, by Thermo Fisher Scientific (4 mentions)
Iscript reagent, by Bio-Rad (4 mentions)
Abgene reagents, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Maxima reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Promega (2 mentions)
Maxima sybr green master mix, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Taqman pcr master 2x reagent, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green jumpstart readymix, by Merck Group (1 mentions)
Sybr greener qpcr supermix, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
Sybr green method, by Thermo Fisher Scientific (1 mentions)
Superscript reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Purelink rna kit, by Thermo Fisher Scientific (1 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
Taqman reagent, by Thermo Fisher Scientific (1 mentions)
17 protocols using icycler
1
Quantification of Nitrogen Cycling Genes
2
Real-time PCR Gene Expression Analysis
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Real-time PCR was performed on the Bio-Rad iCycler with the Faststart SYBR green-based mix (Applied Biosystems). PCR programmes for all genes were designed as follows: 10 min at 95°C, forty cycles at 95°C for 30 s, primer-specific annealing temperature for 30 s and 72°C for 30 s, followed by melting curve analysis. The primer sequences used in this study are listed in Table 3. Primer specificity and efficiency (90-110 %) were verified. Samples were analysed in duplicate, and a difference ≤5 % was acceptable. Relative gene expression was calculated using the 2 ÀΔΔCt method (34) with normalisation against the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase, as described in our previous study (6) .
3
Validating Differential Gene Expression by qRT-PCR
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Six up-regulated and four down-regulated genes were selected to validate the microarray data by qRT-PCR. Primers were designed using the tool primer3 based on the sequence of each differentially expressed probe accessible on the Affymetrix website (Table S3 ). RNA was isolated as previously described, treated with RNase Free DNase Qiagen (Cat #79254) and cleaned with Qiagen RNeasy Mini Kit (P/N 74104). cDNA was synthesized from 1 μg RNA using SuperScriptII Platinum Two-Step qRT-PCR kit (Invitrogen cat # 11735). Quantitative real time PCR (qRT-PCR) was performed in a BioRad iCycler using SYBRGreenER™ qPCR SuperMix (Invitrogen cat# 11761) following manufacturer's instructions. Wheat actin gene was used as internal reference to normalize Ct-values obtained for each gene. qRT-PCR data was analyzed based on Dussault and Pouliot (2006 (link)) method.
4
Gene Expression Analysis of LPS-Induced Inflammation
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Cells were primed with either HMGN1 or LPS and restimulated with LPS as described above. mRNA was extracted by Trizol 4 h post-stimulation. The qPCR primers sequence are listed in the (Table S1 Supplementary Material) For sirtuin-1 expression, cells were stimulated for 4 h before RNA was isolated. cDNA was synthesized from 1 µg of total RNA by use of SuperScript reverse transcriptase (Invitrogen). Relative mRNA levels were determined using the Bio-Rad i-Cycler and the SYBR Green method (Invitrogen). Values are expressed as fold increases in mRNA levels, relative to those in unstimulated cells, with HPRT as a housekeeping gene.
5
Quantification of Arg1 Expression in Murine Muscle
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Real-time PCR was performed as per our published method [11 (link), 12 (link)]. Total RNA was isolated from the quadriceps muscles of mice. The muscle was homogenized and dissolved in TRIzol. RNA was isolated using the TRIzol method following the manufacturer's instructions, and the quality of the RNA preparations was monitored by absorbance at 260 and 280 nm (Helios Gamma, Thermo Spectronic, Rochester, NY). The RNA was reverse-transcribed into complementary deoxyribonucleic acid (cDNA) using iScript reagents from Bio-Rad on a programmable thermal cycler (PCR Sprint, Thermo Electron, Milford, MA). The cDNA (50 ng) was amplified by real-time PCR using a Bio-Rad iCycler and ABgene reagents (Fisher Scientific, Pittsburgh, PA) and ARG1 primers [12 (link)]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control for normalization.
6
Total RNA Isolation from Mouse Tibia
7
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from the frozen tissues using trizol method. The tissues were ground in liquid N 2 with a mortar and pestle, dissolved in Trizol for RNA isolation, per manufacturer's instructions, and the quality of the RNA preparations was monitored by absorbance at 260 and 280 nm (Helios-Gamma, Thermo Spectronic, Rochester, NY). The RNA was then reverse-transcribed into complementary deoxyribonucleic acid (cDNA) using iScript reagents from Bio-Rad on a programmable thermal cycler (PCR-Sprint, Thermo Electron, Milford, MA). 50 ng of cDNA was amplified in each real-time PCR reaction using a Bio-Rad iCycler, ABgene reagents (Fisher scientific), and gene-specific primers (Table 1). Average of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S was used as the internal control for normalization. Standard curves were applied for each RNA assay to produce accurate quantification of the threshold cycle (∆Ct). This allows an approximate comparison of the expression levels of different targets.
8
Quantitative RT-PCR Analysis of Gene Expression
9
Quantitative RT-PCR Analysis of Key Lipid Regulators
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Total RNA from INS-1 cells was extracted from tissues using the Purelink® RNA kit (Thermo Fisher Scientific). Total RNA (1 µg) was reverse transcribed using the SuperScript® Reverse Transcription system (Thermo Fisher Scientific). Real time PCR was performed on the cDNA on a Bio-Rad iCycler using SYBR green mastermix (Thermo Fisher Scientific). Thermal cycling conditions were: 95 °C for 5 min, (30 seconds 95 °C, 30 seconds 60 °C, 1 minute 52 °C) for 40 cycles, followed by a melt curve. Gene expression was expressed as the ratio between the expression of the target gene and GAPDH using the ΔΔCt method. Primers (Integrated DNA Technologies) used: Gapdh (forward 5′-actcccactcttccaccttc-3′, reverse 5′-tcttgctcagtgtccttgc-3′), Abca1 (forward 5′-aacagtttgtggcccttttg-3′, reverse 5′-agttccaggctggggtactt-3′), Ldlr (forward 5′-cagcctagaggggtaaactg-3′, reverse 5′-tagcataccatcagggcaag-3′), Srebf-2 (forward 5′-cgaactgggcgatggatgag-3′, reverse 5′-gacaaactgtagcatctcgtcg-3′).
10
RNA Isolation and Quantification Protocol
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