Total RNA extraction was conducted with the total RNA Miniprep Kit (Axygen Biosciences, Union City, CA, United States). Approximately 5 μg of RNA was reverse transcribed with the ReverTra Ace qPCR-RT Kit (Toyobo, Japan) according to the manufacturer’s protocol. qRT-PCR was carried out in triplicate using the iCycler iQTM Real-time PCR Detection System (Bio-Rad, Hercules, CA, United States) with the procedures as previously described (Yang et al., 2019 (link)). The pepper actin gene was used as an internal control. The primers of actin and CaPIF genes are listed in Supplementary Table 1 .
Icycler iqtm real time pcr detection system
Manufactured by Bio-Rad
Sourced in United States, Japan
The iCycler iQ Real-Time PCR Detection System is a laboratory instrument designed for real-time polymerase chain reaction (PCR) analysis. It provides precise temperature control and optical detection capabilities to quantify and analyze nucleic acid samples in real-time during the amplification process. The system is capable of performing a wide range of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Revertra ace qpcr rt kit, by Toyobo (10 mentions)
Sybr green pcr master mix, by Takara Bio (8 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Sybr green, by Toyobo (4 mentions)
Trizol reagent, by Sangon (4 mentions)
Iq supermix, by Bio-Rad (4 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Icycler software, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trifast reagent, by Avantor (2 mentions)
Iqtm sybr green supermix, by Bio-Rad (2 mentions)
Oligo dt 12 18 primer, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Miniprep kit, by Corning (1 mentions)
Primer express software, by Thermo Fisher Scientific (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Total rna miniprep kit, by Corning (1 mentions)
36 protocols using icycler iqtm real time pcr detection system
1
Quantification of Pepper Gene Expression
2
Quantitative PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cDNAs which were amplified by PCR (previously mentioned) were used in quantitative PCR (qPCR) using a iQ SYBR Green Supermix (Biorad). The amplification of the cDNA was conducted in 35 cycles of: 1) heating at 94°C for 10 min, 2) at 95°C for 10 s, 55°C for 10 s and 72°C for 20 s, and 3) 72°C for 10 min using a iCycler iQtm Real-Time PCR Detection System (Biorad). The comparative Ct method was used for relative measurement of gene expression level against β-actin gene.[29 (link)]
3
Transcriptomic Analysis of Adipocyte Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3T3-L1 cells were seeded into 6-well plates and treated with EJF according to the experimental design to induce differentiation, after which they were washed once with PBS. Total RNA was isolated using Trizol reagent, and then cDNA libraries were synthesized using the PrimeScriptTM RT Reagent Kit (TaKaRa Bio, Kyoto, Japan). For the synthesis of the cDNA library, standard PCR was performed with the following settings: pre-incubation (15 min at 37 °C), annealing (5 min at 50 °C), extension (5 min at 98 °C), and cooling (4 °C). The mRNA expression levels were measured and analyzed using the iCycleriQTM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA), with detection performed using SYBR Green (TOYOBO, Japan). The specific thermal cycling conditions used for RT-PCR were as follows: pre-incubation (1 min at 95 °C), amplification (15 s at 95 °C, followed by 1 min at 60 °C for 39 cycles), melting (10 s at 95 °C), and cooling (5 s at 72.5 °C). The mRNA expression levels were normalized to those of β-actin and indicated as fold changes compared with a control group. All experiments were performed biologically and technically in triplicate. The primers used for RT-PCR were synthesized by Macrogen (Seoul, Republic of Korea), and their sequences are listed in Table 1 .
4
Quantitative Real-Time PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extraction of total RNA and quantitative real-time PCR were performed in a BioRad iCycler iQTM Real-Time PCR detection system using FAM fluorophore-labeled 2X iQ SYBR Supermix (BioRad, Cat# 1708880) as described (Maloney et al., 2009 (link); Rajagopalan et al., 2010 (link)). The threshold cycle (Ct) value of each gene of interest was normalized to the Ct value of 16S rRNA, and the fold expression was calculated [fold change = 2-Δ(ΔCt)]. Expression data were obtained from an average of three independent RNA preparations, and each gene of interest was investigated in triplicate. Fold differences of 2 or more were considered significant. Primers used for qRT-PCR are listed in Supplementary Table S1 .
5
Quantitative Analysis of Mycorrhizal 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 young leaves of both mycorrhizal and non-mycorrhizal plants using Trizol reagent (Sangon, China) according to the manufacturer’s instruction. Genomic DNA was removed with RNeasy Mini Kit (Qiagen, Germany). Total RNA (1 μg) was reverse-transcribed using ReverTra Ace qPCR RT Kit (Toyobo, Japan) following the manufacturer’s instruction. Quantitative real-time PCR was performed using the iCycler iQTM real-time PCR detection system (Bio-Rad, Hercules, CA, United States). Each reaction (25 μL) consists of 12.5 μL SYBR Green PCR Master Mix (Takara, Japan), 1 μl of diluted cDNA and 0.1 μmol of forward and reserve primers. PCR cycling conditions were as follows: 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 30 s. Actin of cucumber (GenBank AB698859.1) was used as an internal standard. The gene-specific primers used for the amplification were determined on the basis of gene or EST sequences and are listed in Supplementary Table S1 . The quantification of mRNA levels is based on the method of Livak and Schmittgen (2001) (link).
6
Quantitative Real-Time PCR Analysis of Tomato Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from tomato leaves using Trizol reagent (Sangon Co., Shanghai, China), according to the manufacture's recommendations. Genomic DNA was removed with the RNeasy Mini Kit (Qiagen Co., Hilden, Germany). 1 μg RNA was reverse-transcribed using the ReverTra Ace qPCR RT Kit (Toyobo Co., Osaka, Japan), following the manufacturer's instructions. Gene-specific RT-PCR primers were designed based on their cDNA sequences (Supplemental Table S1 ).
The quantitative real-time PCR was performed using the iCycleri QTM real-time PCR detection system (Bio-Rad Co., Hercules, CA, USA). Each reaction (25 μL) consisted 12.5 μL of SYBR Green PCR Master Mix (Takara Co., Chiga, Japan), 1 μL of diluted cDNA and 0.1 μmol forward and reserve primers. The PCR cycling conditions and the calculation of relative gene expression were as previously described. The tomato ACTIN gene was used as internal control as previously described (Zhou et al., 2014a (link)).
The quantitative real-time PCR was performed using the iCycleri QTM real-time PCR detection system (Bio-Rad Co., Hercules, CA, USA). Each reaction (25 μL) consisted 12.5 μL of SYBR Green PCR Master Mix (Takara Co., Chiga, Japan), 1 μL of diluted cDNA and 0.1 μmol forward and reserve primers. The PCR cycling conditions and the calculation of relative gene expression were as previously described. The tomato ACTIN gene was used as internal control as previously described (Zhou et al., 2014a (link)).
7
Quantitative Real-Time PCR for Virus Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative real-time PCR (qPCR) was carried out using iCycler iQTM Real-Time PCR Detection System (BioRad Laboratories, Hercules, CA, USA), as described previously [22 (link)], with the following cycling parameters: 1 cycle at 50 °C for 3 min; 1 cycle at 95 °C for 5 min; 45 cycles, each consisting of 15 s at 95 °C and 1 min at 60 °C. A melting curve was recorded at the end of each run to assess amplification specificity. All reactions were performed with three technical replicates. PCR efficiency was calculated using standard curves constructed with serial dilutions of DNA extracted from infected plants. Data acquisition and analysis were handled by the BioRad iCycler software (version 3.06070) that calculates Ct values and standard curves. The primer pair TY2222(+)/TY2371(−) (Table 1 ) was used to amplify TYLCSV genomic fragments, while the primer pair SlyAPX-862(+)/SlyAPX-948(−) (Table 1 ) was used for the amplification of the tomato gene Y16773.1 coding for ascorbate peroxidase (APX), utilized as reference gene. The relative virus amount was estimated according to [20 (link)].
8
Gene Expression Analysis of Amnion Mesenchymal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from amnion mesenchymal cells using Trizol, isolated using the RNeasy Mini-Kit and RNA concentrations were quantified using the NanoDrop® spectrophotometer. For each sample, 0.5–1.0 μg of RNA was reversed transcribed into cDNA using the Superscript III® first strand system (Thermo Fisher Scientific). Twenty-five to fifty nanograms of cDNA were used as the template for each real-time PCR reaction. Real-time PCR was performed using pre-validated Taqman probes directed against MMP1 (assay ID: Hs00899658_m1) and GR (NR3C1) (Assay ID:Hs00353740_m1). Forward and reverse primers were used to detect PGRMC1, IL8 mRNA and the housekeeping gene B2M mRNA expression (Table 1 ). We performed Real-Time PCR using the following protocol: initial denaturation at 95°C for 3 min, followed by a 2-step amplification process of 95°C for 30 s and 60°C for 40 s for a total of 40 cycles. Real-Time PCR was performed using the iCycler IQTM Real-Time PCR detection system (Bio-Rad). All samples were run in duplicates with the mean cycle threshold Ct for the gene of interest normalized to the mean Ct value for the housekeeping gene B2M.
9
Quantifying Gene Expression in Cucumber Leaves
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from grafted cucumber leaves using an RNA extraction kit (Axgen, Union City, CA, USA) according to the supplier’s instructions. DNA contamination was removed using a purifying column. One microgram of total RNA was reverse-transcribed using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) following the supplier’s recommendations. The gene-specific primers for qRT-PCR were designed based on their cDNA sequences, as follows: RBCL (F, 5′-ATTTGCGAATCCCTACT-3′; R, 3′-AAACCGCTCTACCATAA-5′), RBCS (F, 5′-ACCACAGGTCACCAGGAT-3′; R, 3′-GGGCTTGTAGGCGATG-5′), RCA (F, 5′-AAGTGAGAAAGTGGGCTGTA-3′; R, 3′-TTGTCATCTTCGGTTGGT-5′), and the actin gene (F, 5′-TGGACTCTGGTGATGGTGTTA-3′; R, 3′-CAATGAGGGATGGCTGGAAAA-5′) was used as an internal control. The qRT-PCR assays were performed using an iCycleriQTM Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). PCRs were performed using the SYBR Green PCR Master Mix (Takara, Tokyo, Japan). The PCR conditions consisted of denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 30 s. The quantification of mRNA levels was based on the method of Livak and Schmittgen (2001) (link).
10
Barley Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pot experiment was carried out again using XZ5, XZ54 and cv. ZAU3 under control and drought stress treatment with three replicates as described above. Total RNA was isolated from leaves of barley plants under control and SMC 10 % and 4 % using the TRIzol reagent following manufacturers’ recommendation (Invitrogen, Karlsruhe, Germany). Residual DNA was removed using purifying columns. One microgram of each RNA sample was subsequently employed for cDNA synthesis with 0.5 μg of oligo (dT) 12-18 and 200 units of Superscript II (Invitrogen, Karlsruhe, Germany). cDNA samples were assayed by quantitative real time PCR (qRT-PCR) in the iCycler iQTM Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the SYBR Green PCR Master Mix (Applied Biosystems). The PCR conditions consisted of denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s. Gene-specific primers (Additional file 1 : Table S1) were designed using the Primer Express software (Applied Biosystems). Barley GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene (accession no. M36650, fw-5’-AAGCATGAAGATACAGGGAGTGTG-3’, rv-5’-AAATTTATTCTCGGAAGAGGTTGTACA-3’) and barley ACTIN (AY145451, fw-5’-ATGTTTTTTTCCAGACG-3’, rv- 5’-ATCAAGCCAACCCAAGT-3) were used as control.
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!