Gene expression was measured on an iQ5 qPCR system (Bio-Rad) using SYBR Green qPCR master mix (Bimake, catalog no. B21202) or iTaq Universal Probes Supermix (Bio-Rad, catalog no. 1725130). CT values were defined at the inflection points of fitted sigmoid curves (four-parameter Chapman curves) and were compared with mitochondrial processing peptidase (Pmpca).
Iq5 qpcr system
Manufactured by Bio-Rad
Sourced in United States
The IQ5 qPCR system is a real-time PCR detection system designed for quantitative gene expression analysis. It features a 96-well format, a high-resolution CCD camera, and software for data analysis and management.
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Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Rneasy kit, by Qiagen (2 mentions)
Sybr green qpcr master mix, by Selleck Chemicals (1 mentions)
Itaq universal probes supermix, by Bio-Rad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Maxima first stand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Bio-Rad (1 mentions)
Gapdh, by Bio-Rad (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Miracloth, by Avantor (1 mentions)
Genejet plant genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
Maxima sybr green qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Dnase 1 treatment, by Qiagen (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
12 protocols using iq5 qpcr system
1
qPCR Gene Expression Analysis
2
Quantifying Fungal Protease Gene Expression
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Specific PCR primers targeting C. rosea S8A serine protease genes were designed using the PrimerSelect software implemented in DNASTAR Lasergene ver. 10 package (DNASTAR Inc., Madison, WI). Primer amplification efficiencies were determined based on amplification of serial dilutions of C. rosea genomic DNA or cDNA. Transcript levels were quantified by an iQ5 qPCR system (Bio-Rad, Hercules, CA) using the SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA) as described previously [77 (link)]. After qPCR reactions, melt curve analyses were performed in order to confirm that the signal was the result of a single product amplification. The relative expression levels for protease genes were calculated in relation to the reference gene tubulin [78 (link)] by using the 2–ΔΔCT method [79 (link)]. Gene expression analysis was performed in five biological replicates, each based on two technical replicates.
3
RT-qPCR Protocol for Gene Expression Analysis
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RNA extraction was performed using the Qiagen RNeasy kit following the manufacturer's protocol (Qiagen). After DNaseI (Fermentas, St. Leon‐Rot, Germany) treatment, one microgram of total RNA was reverse transcribed in a total volume of 20 μl using Maxima first stand cDNA synthesis kit (Fermentas, St. Leon‐Rot, Germany). Transcript levels were quantified by RT‐qPCR using the SYBR Green PCR Master Mix (Fermentas, St. Leon‐Rot, Germany) and primer pairs listed in Appendix S1 , in an iQ5 qPCR System (Bio‐Rad, Hercules, CA, USA) as described previously (Tzelepis, Melin, Jensen, Stenlid, & Karlsson, 2012 ). Melt curve analysis was performed after the qPCR reactions, to confirm that the signal was the result from a single product amplification. Relative expression levels for target genes in relation to actin (act), shown previously to be constitutively expressed (Kamou et al., 2016 ; Tzelepis, Dubey, Jensen, & Karlsson, 2015 ), were calculated from the Ct values by the 2 − Δ Δ C t 2001 ). Gene expression analysis was carried out in at least three biological replicates, each based on two technical replicates. Gene expression data were analysed by analysis of variance (ANOVA) using a general linear model approach implemented in Minitab ver. 18 (Minitab Inc., State College, PA, USA). Pairwise comparisons were made using the Fisher method at the 95% significance level.
4
Quantitative Analysis of Foxp3 Expression
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The reverse transcription kit and qPCR kit were purchased from Dalian Takara Company. The total RNA of spleen lymphocytes in each group was extracted by the TRIzol method. Total RNA concentration was determined by spectrophotometry. A cDNA template was obtained by using reverse transcription kit to extract 500 ng of total RNA from mouse lymphocytes. qPCR primers for target genes were designed and synthesized by Shanghai Sangon Co., Ltd.: Foxp3 Sense: 5′-CACCTATGCCACCCTTATCCG-3′, Foxp3 Anti-sense: 5′-CATGCGAGTAAACCAATGGTAGA-3′; GAPDH Sense: 5′-AGGTCGGTGAACGGATTTG-3′, GAPDH Anti-Sense: 5′-GGGGTCGTTGATGGCAACA-3′. Procedures were performed as described in SYBR Premix Ex TaqTM II and tested by the U.S. Bio-Rad iQ5 qPCR system. Reaction conditions are as follows: 94°C, 30 s; 58°C, 30 s; and 72°C, 30 s, with a total of 40 cycles. As an internal reference gene, GAPDH was repeated for 3 times, and the mRNA expression of each group was calculated by the 2-ΔΔCT method.
5
Quantifying Fungal Gene Expression
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Total RNA was extracted from 1-week-old cultures of wild-type (Guy 11), ΔCyp1 and ΔCyp1+ M. oryzae strains using the RNeasy Plant Mini Kit (Qiagen) according to manufacturer’s instructions and concentrations were determined using NanoDrop (Thermo Scientific). For cDNA synthesis, 1 µg total RNA was reversed transcribed in a total volume of 20 µl using the iScript cDNA Synthesis Kit (Bio-Rad). Transcript levels were quantified by quantitative reverse transcriptase PCR (RT-qPCR) using the iQ5 qPCR System (Bio-Rad) as described previously (Tzelepis et al. 2012 (link)). Relative expression values of PbCYP3 gene were calculated using the 2−ΔΔCT method (Livak and Schmittgen 2001 (link)). The M. oryzae actin gene (Che Omar et al. 2016 (link)) was used to normalize the data using the MgActF/R primers listed in Table 1 .
6
Quantifying Alternaria Infection in Plants
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For necrotrophic fungal infection, Alternaria brassicicola strain MUCL20297 was cultured on potato dextrose agar plates for 2 weeks at 22 °C. Spores were harvested in water and filtered through Miracloth (EM475855-1R, VWR) to remove hyphae. The spore suspension was adjusted to the final concentration of 5 × 105 spores ml–1 supplemented with 0.05% Tween-20. Alternaria brassicicola inoculation of 3-week-old plants was performed by adding 10 µl drops of spore suspension onto the upper leaf surface as described previously (Thomma et al., 1998 ). Plants were maintained under saturating humidity for 1 d prior to pathogen inoculation and 2 d post-inoculation. Leaf samples for fungal quantification were collected 7 d post-inoculation, snap-frozen in liquid nitrogen, and stored at –70 °C prior to DNA extraction. Total DNA was extracted from frozen leaf samples using the GeneJET Plant Genomic DNA Purification Kit (K0791, Thermo Fisher Scientific) following the manufacturer’s protocol. Fungal DNA quantification of three independent biological replicates was carried out by quantitative real-time (qRT)-PCR using the iQ5 qPCR System (Bio-Rad) to detect fungal cutinase (GI 416217) and Arabidopsis UBQ5 (AT3G62250) and PR2 (AT3G57260) genes with corresponding primer pairs listed in Supplementary Table S5 .
7
Quantifying Gene Expression in Developing Arabidopsis Seeds
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Ovules/seeds of 25 emasculated or hand-pollinated WT siliques were harvested at 2DAE, 1, 2, 3, 4 and 5 DAP in 20 μL of RNAlater solution (Invitrogen) and ground for 2 min using a TissueLyser II (Qiagen AB, Sweden). Total RNA was extracted using the Qiagen RNeasy kit, followed by DNase I treatment (Qiagen). cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Fisher Scientific, Sweden). Maxima SYBR Green qPCR Master Mix (Thermo Scientific) was used to perform the qPCR in an iQ5 qPCR system (Bio-Rad Laboratories AB, Sweden). The primers used for the RT-qPCR are described in Table 2 . PP2A was used as the reference gene. Relative quantification of gene expression was performed as described (Pfaffl, 2001 (link)). Expression levels for each gene were normalized to the expression level at 2 DAE.
8
miRNA Quantification from Cells and Tissues
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Total RNA was extracted from cells and tissues using Trizol (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer′s instructions. The RNA was quantified by absorbance at 260 nm. To assess the levels of miRNAs, qRT‐PCR analysis was conducted by using Taqman probes (Invitrogen) in an IQ5 Q‐PCR system (Bio‐Rad, Hercules, CA, USA) in accordance with the manufacturer′s instruction. Primers for qRT‐PCR are shown in Tables S2 and S3 .
9
Quantifying mRNA Expression in Cancer Cells via qPCR
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Real-time polymerase
chain reaction (qPCR) was used to quantify mRNA expression in cancer
and normal cells. The assay was conducted using Power SYBR Green Cells-to-Ct
Kit (Invitrogen). Cells were lysed using RNA lysis solution (Invitrogen).
RNA lysates were reverse transcribed to synthesize cDNA. Primers for
qPCR were designed using freely available software from IDT DNA. Identified
primers were purchased from IDT DNA and tested for the amplification
of a single uniform amplicon through analysis of SYBR melting curves
for two cell lines (HeLa and A-549). All qPCR reactions were performed
in 20 μL reaction mixtures. Each sample was run in triplicate
with the IQ5 qPCR system (Bio-Rad). Each plate probed the expression
of Bcl-2 and ACTB genes in the target cell lines. Real-time PCR data
were analyzed using the comparative CT method, also known
as the 2–ΔΔCT method, in
which the expression of Bcl-2 mRNA was normalized to the expression
of the housekeeping gene ACTB.
chain reaction (qPCR) was used to quantify mRNA expression in cancer
and normal cells. The assay was conducted using Power SYBR Green Cells-to-Ct
Kit (Invitrogen). Cells were lysed using RNA lysis solution (Invitrogen).
RNA lysates were reverse transcribed to synthesize cDNA. Primers for
qPCR were designed using freely available software from IDT DNA. Identified
primers were purchased from IDT DNA and tested for the amplification
of a single uniform amplicon through analysis of SYBR melting curves
for two cell lines (HeLa and A-549). All qPCR reactions were performed
in 20 μL reaction mixtures. Each sample was run in triplicate
with the IQ5 qPCR system (Bio-Rad). Each plate probed the expression
of Bcl-2 and ACTB genes in the target cell lines. Real-time PCR data
were analyzed using the comparative CT method, also known
as the 2–ΔΔCT method, in
which the expression of Bcl-2 mRNA was normalized to the expression
of the housekeeping gene ACTB.
10
Quantifying gene expression from iN lysates
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RNA from iN lysates was isolated and collected using Quick-RNA MiniPrep Kit (Zymo Research, R1054). 2 μg of RNA per sample was treated with 2 U of TURBO DNase (Thermo Fisher Scientific, AM2238) for 30 min at 37°C twice to remove contaminating genomic and plasmid DNA, and then recovered using the RNA Clean & Concentrator-5 Kit (Zymo Research, R1015). cDNA from each sample was generated from 250 ng of RNA from the previous step with a mixture of oligo(dT) and random hexamer primers (iScript cDNA Synthesis Kit, Bio-Rad, 1708891). Finally, cDNA abundance was measured using iQ SYBR Green Supermix (Bio-Rad, 1708882) from an iQ5 qPCR system (Bio-Rad), and the appropriate primers at 100 nM. Primer information is listed in Table 7 . cDNA abundance was quantified using a modified ΔΔCt method recommended by the manufacturer.
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