RNA (500 μg, same material used for RNA sequencing) was reverse transcribed into cDNA and subsequently used as a template for quantitative real‐time PCR (qRT‐PCR) as described previously (Poliner et al., 2015 (link)). Primers were designed using IDT PrimerQuest (Integrated DNA Technologies, Coralville, IA) to amplify regions that are conserved between all Atlantic gene isoforms and to span one exon‐exon boundary within the gene for all intron‐containing genes (Table S6 ). Relative expression was determined using the comparative CT method with the average CT values of Soltu.DM.06G026290 and Soltu.DM.03G006780 as an internal control.
Idt primerquest
Manufactured by Integrated DNA Technologies
Sourced in United States
IDT PrimerQuest is a web-based tool that assists in the design of DNA primers and probes. It uses proprietary algorithms to generate optimal primer and probe sequences based on user-provided input sequences. The tool helps researchers streamline the primer design process for a variety of applications, such as PCR, qPCR, and DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
2200 tapestation system, by Agilent Technologies (1 mentions)
D1000 screentape, by Agilent Technologies (1 mentions)
Sybr green 2 step qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
Dynamotm cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rotor gene q software, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Netprimer, by Premier Biosoft (1 mentions)
Icycler, by Bio-Rad (1 mentions)
4 protocols using idt primerquest
1
Quantitative real-time PCR analysis
2
Sanger Sequencing of ASXL1 Variant
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Sanger sequencing was performed on a recurrent ASXL1 c.1934dup G646WfsTer12 variant detected in 75% of the samples to verify if the variants with VAF < 5% were genuinely sequencing errors in this study (Supplementary S1 : ASXL1 NM_015338.5:c.1934dup (VAF < 5%) validation, Supplementary File S1 ). The IDT Primer Quest (Integrated DNA Technologies, Inc. was used to design the primers for the variant of interest (ASXL1c.1934dup) and checked for specificity using the NCBI Primer-BLAST. Optimisation of the annealing temperature DNA input was performed, followed by mastermix preparation and PCR cycles. The PCR thermocycling protocols were as follows: 98 °C for 10 s for initial denaturation, 60 °C for 5 s and 72 °C for 15 s (30 cycles), followed by a final extension at 72 °C for 1 min. The PCR products were visualised using Agilent’s D1000 Screentape with Agilent’s 2200 TapeStation system. PCR product purification was performed using Geneall Exspin PCR SV (103-102) according to the manufacturer’s instructions, and the samples were assessed for purity using the Nanodrop spectrophotometer. Samples with acceptable concentration and purity ratios were sent for Sanger sequencing. The Sanger sequencing data were reviewed using the Chromas software (version 2.6.6) (C. McCarthy; accessed on 20 January 2022), http://www.technelysium.com.au/chromas2.html ).
3
Quantitative Analysis of Lhb Gene Expression
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Total RNA was extracted using the TRIZOL reagent (Invitrogen, USA) following the manufacturer 's protocol. cDNA was synthesized from 800 ng of the total RNA using anchored random hexamer primers and Moloney murine leukaemia virus reverse transcriptase according to the protocol of DyNAmo TM cDNA synthesis kit (Finnzymes, Finland). Real-time PCR amplification was carried out using SYBRGreen 2-step qRT-PCR kit (Finnzymes, Finland) according to the manufacturer's protocol. Relative Lhb gene expression was calculated using the comparative quantitation option of Rotorgene-Q software (Qiagen, USA). To compensate for the variation in cDNA concentrations and the PCR efficiency between tubes and endogenous control, the rat glyceraldehyde-3-phosphate dehydrogenase (rGapdh) gene was quantified in each sample and used for normalization. The average relative Lhb gene expression of the control group was set to 1.0. Pairs of primers specific for the rLhb gene and primers specific for the reference rGapdh gene were designed to span over intron sequences using the Primer3 open-source software [17] (link) (IDT PrimerQuest; Integrated DNA Technologies Inc., USA). Primer specificity was confirmed by a BLAST software-assisted search of a nonredundant nucleotide sequence database (National Library of Medicine, USA). Specific primer sequences were as follows:
4
Quantifying Gene Expression using qPCR
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Primers for real-time qPCR were made to ACTIN2, PIF1, and CTG10 (Table S1 ) cDNA using IDT PrimerQuest (Integrated DNA Technologies, Inc.) and the Primer3 program (93 (link), 94 (link)) and were analyzed using NetPrimer (PREMIER Biosoft; NetPrimer/" xlink:show="new">www.premierbiosoft.com/NetPrimer/ ). A Bio-Rad Laboratories iCycler permitted measurement of SYBR Green fluorescence to determine abundance of the PIF1 and CTG10 transcripts relative to ACTIN2.
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