Total RNA was collected from cultured cells using High Pure RNA Isolation Kit (Roche Diagnostics Corporation). Total RNA was collected from mouse tissue using TRIzol Reagent (Ambion Life Technologies) followed by RNeasy clean-up with DNAse treatment (Qiagen) according to the manufacturer's instructions. Total RNA and purity was assessed using a nanospectrometer (Thermo Scientific NanoDrop Lite). cDNA was synthesized from 500 ng of RNA using a reverse transcription kit, TaqMan® Reverse Transcription Reagents (Thermo Fisher Scientific) and Oligo(dT) primer. Aliquots of cDNA were added to a reaction mixture containing 2X Universal SYBR® Green Supermix (Bio-Rad) and 400 nM primers. Target quantity was analyzed against a standard curve, using a ViiA 7 instrument and software (Applied Biosystems) and normalized to β-actin. The absence of DNA contamination was confirmed by carrying out amplification from cDNA without reverse transcriptase. The primers were designed with IDT PrimerQuest software (Integrated DNA Technologies, Inc.).
Idt primerquest software
Manufactured by Integrated DNA Technologies
Sourced in United States
IDT PrimerQuest software is a bioinformatics tool designed for the identification and analysis of DNA primer sequences. The software's core function is to assist researchers in the selection of optimal oligonucleotide primers for use in various molecular biology techniques, such as PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
High pure rna isolation kit, by Roche (2 mentions)
Nanodrop lite, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Bio-Rad (1 mentions)
Viia7 cycler, by Thermo Fisher Scientific (1 mentions)
Prism v6, by GraphPad (1 mentions)
Spss version 22, by IBM (1 mentions)
Imagej software, by Oxford Instruments (1 mentions)
3500 instrument model, by Thermo Fisher Scientific (1 mentions)
4 protocols using idt primerquest software
1
RNA Isolation and Real-Time qPCR Protocol
2
Quantifying Gene Expression by qRT-PCR
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qRT-PCR was performed essentially as described [69 (link)], except that RNA was isolated and purified using the High Pure RNA Isolation Kit (Roche Diagnostics) and RT-qPCR was carried out using 2X SYBR® Green PCR Master Mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in a ViiA7 cycler (Applied Biosystems, Inc., Foster City, CA, USA). Primers for PCR were designed with IDT PrimerQuest software (Integrated DNA Technologies, Inc., Coralville, IA, USA): DCYTB forward 5′-TGCATACAGTACATTCCCGCCAGA-3′, DCYTB reverse 5′-ATGGAACCTCTTGCTCCCTGTTCA-3′, ACTB forward 5′-TTGCCGACAGGATGCAGAAGGA-3′, ACTB reverse 5′-AGGTGGACAGCGAGGCCAGGAT-3′. GREB1 primers were as described in [70 (link)].
3
Quantification of ER Stress and Apoptosis
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PCR primers were configured using IDT PrimerQuest software (Integrated DNA Technologies, Coralville, IA, USA). Image‐based data were resolved using ImageJ software (Oxford Instruments, Abingdon, UK). The statistical significance was explored using GraphPad Prism V6 software (GraphPad Software Inc., La Jolla, CA, USA). Expression results were calculated using the 2ΔΔct method. All technical and biological experiments were performed with at least three replicates (N ≥ 3). The SPSS version 22.0 (IBM Corp., Armonk, NY, USA) was used to analyze the study data. The data were first examined for a normal distribution based on skewness and kurtosis values (which were between 0.846 and 0.924) and Q_Q plot graphics. The data showed a normal distribution. Based on these results, an independent group t‐test was used to compare the ER stress and apoptosis protein levels in the CA and AH groups. The level of significance was defined as p < .05.
4
Molecular Characterization of Staphylococcal agr Loci
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Primers for agrA and agrBD were designed using online idt primer quest™ software of Integrated DNA Technologies, USA (www.idtdna.com ) using SP ED99 strain and HKU10-03 strain agr gene sequences available in the NCBI database (Accession No. CP002478 and CP002439, respectively). Then, the selected primers were BLAST searched for the specificity using Blastn program of NCBI. Amplification of agr genes were performed as mentioned above, and purification of the amplicon was done using PCR purification kit (Real Biotech Corporation, Taiwan) as per the manufacturer’s instruction. DNA sequencing of purified nucleotides was done with automated high throughput nucleic acid sequence of Applied Biosystems 3500 Instrument model. Homology searches were performed with the NCBI database and BLAST. Alignment and phylogenetic tree analysis were carried out using the Mega5.2 software. In silico amino acid analysis was done using DNAstar software.
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