Total RNA from Arabidopsis leaves was extracted using Tri-sure (Bioline) and subsequently purified using the RNA Clean and Concentrator-5 kit (Zymo Research). RNA samples were treated with NZY DNase I (NZYTech). First-strand cDNA was synthesized from 1 μg of purified total RNA by using the PrimeScript RT Master Mix (Takara). Real-time quantitative PCR (RT–qPCR) reactions were performed using SYBR®Premix Ex TaqTM (Takara) and an iCycler 5 (Bio-Rad). All kits were used according to the manufacturers’ instructions. Relative quantification of specific mRNA levels was performed using the comparative 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)) by using the gene-specific primers described in Supplementary Table S1 . Expression values were normalized using the Arabidopsis housekeeping genes TUBULIN-4 (At5g44340) or ACTIN7 (At5g09810). Fungal infection was measured by analysing the B. cinerea β-TUBULIN gene (XM_001560987.1) relative to the Arabidopsis TUBULIN-4 gene.
Icycler5
Manufactured by Bio-Rad
Sourced in United States
The iCycler5 is a programmable thermal cycler designed for nucleic acid amplification and analysis. It provides precise temperature control and automated cycling for PCR and other thermally-sensitive processes. The device features a user-friendly interface and is suitable for a range of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green, by Quanta Biosciences (2 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (2 mentions)
Random hexamer, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nzy dnase 1, by NZYTech (1 mentions)
Trisure, by Meridian Bioscience (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Sybr premix ex taqtm, by Takara Bio (1 mentions)
Rna clean and concentrator 5 kit, by Zymo Research (1 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Prometheus system, by NanoTemper (1 mentions)
Oligonucleotide primers, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Faststart sybr green master mix, by Roche (1 mentions)
15 protocols using icycler5
1
Quantification of Arabidopsis Gene Expression
2
Quantitative Real-Time PCR Analysis of Mtb
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested from two biological replicates of all Mtb strains and RNA was isolated as described previously [58 (link)]. First-strand synthesis was performed by using 500 ng total RNA with iScript Select cDNA synthesis kit (Bio-Rad) using random oligonucleotides. PCR was performed in three technical replicates using gene specific primers. Expression of genes was analyzed with real-time PCR using iQ SYBR Green supermix (Bio-Rad) and a BioRad iCycler 5 with an iQ Multicolour Real-Time PCR Detection System (Bio-Rad). PCR efficiencies were normalized to obtain accurate expression levels. For comparisons between wt Mtb and MtbΔWhiB3, the induction ratio for each gene was normalized to Mtb 16s rRNA expression.
3
RNA Isolation, RT-qPCR, and mRNA Stability
4
Measuring Protein Thermal Stability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two separate methods were used to measure the melting temperature (Tm) of the purified PAS domain. One method was based on Nano differential scanning fluorimetry (NanoDSF)44 (link) and used the Prometheus system from Nanotemper. The latter system recorded the intrinsic tryptophan and tyrosine fluorescence. The ratio of the fluorescence intensities at 350 nm and 330 nm was determined while the temperature was steadily increased from 20 to 95 °C which results in a melting curve. The inflection point of the melting curve is considered as the Tm. Ligand binding was analyzed by determining the impact of potential ligands on the Tm value. As a second method, the fluorescent dye SYPRO orange was added to the protein and the fluorescent signal was measured in a real-time PCR instrument (Bio-Rad iCycler5) while the temperature was steadily increased from 10 to 80 °C. The dye binds preferentially to hydrophobic regions resulting in an increase in fluorescence emission while the protein unfolds and hydrophobic parts become exposed45 (link),46 (link). The ∆ Tm is calculated by comparing the Tm of the respective sample to a control without ligand.
5
Gene Expression Analysis of Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA from breast cancer cells was extracted according to the manufacturer's instructions (Qiagen, Valencia, CA) and cDNA was generated with qScript cDNA synthesis kit (Quanta BioSciences, Gaithersburg, MD), followed by qPCR using SYBR green (Quanta BioSciences, Gaithersburg, MD) on an iCycler5 (Bio-Rad, Hercules, CA). Amplification of 36B4, a housekeeping gene, was used for normalizing gene expression values.
6
RNA Extraction and qPCR Analysis of Islet Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from islets of Langerhans from both experimental groups using TRIzol Reagent (Gibco-BRL, Rockville, MD, USA). Integrity of the obtained RNA was verified by agarose–formaldehyde gel electrophoresis. Cross-contamination with protein or phenol was checked by measuring the 260:280 nm absorbance ratio. Samples with a ratio of 1.8 or lower were discarded. On the other hand, samples were treated with DNase I (Invitrogen, Waltham, MA, USA) to avoid DNA contamination. Reverse transcription-PCR was conducted with SuperScript III Reverse Transcriptase (Invitrogen), oligo-dT and 1 μg of total RNA as template. qPCRs were run in triplicate using FastStart SYBR Green Master mix (Roche) in the iCycler 5 (BioRad) employing 40 cycles (denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s). Every run was followed by a melting curve from 55 °C to 90 °C for checking the presence of only one amplicon. Sequences of oligonucleotide primers (Invitrogen) used in these experiments are shown in Table 2 . Quantified values were normalized using β-actin as a housekeeping gene employing individual efficiency calculated with a standard curve for each gene.
7
Evaluating apoptotic and proliferative genes
8
qPCR Analysis of Wnt Pathway Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted according to the manufacturer's instructions (Qiagen, Valencia, CA, USA), and cDNA was manufactured using qScript cDNA synthesis kit (Quanta BioSciences, Gaithersburg, MD, USA), followed by qPCR using SYBR green (Quanta BioSciences) on an iCycler5 (Bio-Rad, Hercules, CA, USA). Amplification of 36B4, a housekeeping gene, was used for normalizing gene expression values. Primer sequences were as follows: DDX3 F 5′-GGAGGAAGTACAGC CAGCAAAG-3′, DDX3 R 5′-CTGCCAATGCCATCGTAATCACTC-3′, Axin-2 F 5′-TCAAGTGCAAACTTTCGCCAACC-3′, Axin-2 R 5′-TAGCCAGAACCTATGTGATAAGG-3′, c-Myc F 5′-CGTCTCCACACATCAGCACAA-3′, c-Myc R 5′-CACTGTCCAACTTGACCCTCTTG-3′, Cyclin D1 F 5′-GGCGGAGGAGAACAAACAGA-3′, Cyclin D1 R 5′-TGGCACAGAGGGCAACGA-3′.
9
Quantitative RT-PCR Analysis of mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total mRNA from animal tissues was isolated using TRIzol Reagent (Invitrogen). RNA was reverse-transcribed using The PrimeScript RT Master Mix (Perfect Real Time) (Takara, Dalian, China). Real-time quantitative RT-PCR was performed using the iCycler 5 thermal cycler (BioRad, CA, United States). Each cDNA was amplified in a 20 μl volume using the SYBR Premix EX Taq kit (Takara, Dalian, China), with a 500 nM final concentration of each primer. The amplification specificity was checked using melting curve analysis. For each cDNA, all target gene mRNA levels were normalized to GAPDH mRNA levels. Results are expressed as the ratio of normalized target gene mRNA levels in treated groups relative to those in the saline group.
10
Quantifying Exercise-Induced Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
qPCR was performed using iCycler5 and CFX manager software analyzer (Bio-Rad Laboratories). IL-6, FNDC5, and METRNL primers were custom-designed and purchased from Integrated DNA Technologies (IL-6 forward: 5′ GAC CCA ACC ACA AAT GCC A-3′; reverse: 5′-GTC ATG TCC TGC AGC CAC TG-3′; FNDC5 forward: 5′-AGG TGT CAT TGC CCT CTT CT-3′; reverse: 5′-CTG GTG TGC TGG TTT CTG AT-3′; METRNL: forward 5′-TCC ATC CAG CAA GTT ACC-3′; reverse: 5′-GCT CGA AGA CCC TGC TTT-3′). Forward and reverse primers were diluted separately and combined for the reaction to a final concentration of 5 µmol/L. Beta-actin, which remained stable with acute exercise and training, was utilized as a housekeeping gene. Pooled cDNA from pretraining biopsies of a subject who did not complete the training portion of the study were used to run efficiency curves for IL-6, FNDC5, and METRNL, where all efficiencies were between 90%–110%. qPCR was then performed on a per target basis according to MIQE guidelines.15 (link) Using CFX manager software, the relative amounts of mRNA expression were analyzed using the 2−ΔΔCT method, with mRNA content expressed relative to the resting (baseline) pretraining biopsy.
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!