Extracol reagent

Manufactured by EURx

Sourced in Poland

Extracol is a reagent used in molecular biology and biochemistry laboratories. It is a solution that is used to extract and purify nucleic acids, such as DNA and RNA, from various biological samples. The core function of Extracol is to facilitate the isolation and separation of nucleic acids from other cellular components in a sample.

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RNA Extracol Reagent (2 citations)

4 protocols using extracol reagent

RNA Extraction and RT-qPCR Analysis

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Total RNA from embryos was extracted using the Extracol reagent (EURX, Poland), following the provided protocol. RNA was quantified using the NanoDrop OneC Spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA integrity was confirmed by electrophoresis. The cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, MA, USA). Real-time quantitative PCR (RT-qPCR) was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA) utilizing the PowerUp SYBR Green Master Mix (Thermo Scientific, Waltham, MA, USA) with the gene-specific primers indicated below (Table 1.).
The software automatically determined the Ct values. Standard curves for each pair of primers were prepared by serial 5-fold dilutions of the template cDNA followed by the determination of reaction efficiencies.

Dubińska-Magiera M., Niedbalska-Tarnowska J., Migocka-Patrzałek M., Posyniak E, & Daczewska M. (2020). Characterization of Hspb8 in Zebrafish. Cells, 9(6), 1562.

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CAGE-Seq Transcriptome Profiling Protocol

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Total RNA was isolated using an Extracol reagent per the manufacturer’s protocol (EURX, Gdansk, Poland). The quality of total RNA was assessed by Agilent 2,100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States) to confirm that RNA integrity number is over 8.0. Reverse transcription of RNA was performed using random primers (CAGE library preparation Kit; K.K. DNAFORM, Yokohama, Japan). The selection of RNA/cDNA hybrids was enabled by cap-trapping on streptavidin beads. Following the digestion of RNA using RNaseI/H, the linker ligation to 5′ and 3’ cDNA ends enabled the construction of double-stranded cDNA libraries. Sequencing of libraries using the Cap analysis gene expression (CAGE) method was performed using 75 nt single-end reads on a NextSeq 2000 instrument (Illumina, San Diego, CA, United States). Data from this experiment are deposited in Gene expression omnibus (GEO) database (accession number GSE229210). The quality of the obtained data was evaluated via the FastQC tool (v0.11.9). Reads alignment and mapping to a human reference genome (hg38) were performed using the STAR method (Dobin et al., 2013 (link)). Counting tags at each side was performed using SAMtools in R (Rsamtools v2.2.3). In each cell line, CAGE-Seq was performed in biological triplicate for the “WWOX” and “Vec” cellular variants.

Kałuzińska-Kołat Ż., Kołat D., Kośla K., Płuciennik E, & Bednarek A.K. (2023). Molecular landscapes of glioblastoma cell lines revealed a group of patients that do not benefit from WWOX tumor suppressor expression. Frontiers in Neuroscience, 17, 1260409.

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RNA Extraction and qPCR Analysis

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Total RNA from investigated larvae was extracted using the Extracol reagent (EURX, Gdańsk, Poland), according to the protocol provided by the manufacturer. RNA was quantified using the NanoDrop OneC Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The cDNA was synthesised using a HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems, Bedford, MA, USA).
Real-time quantitative PCR (RT-qPCR) was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) utilising the PowerUp SYBR Green Master Mix (Thermo Scientific, Waltham, MA, USA) with the gene-specific primers indicated below (Table 1).
The software automatically determined the Ct values. Standard curves for each pair of primers were prepared by serial 5-fold dilutions of the template cDNA followed by the determination of reaction efficiencies. The number of atrogin-1 molecules was determined from the curve and then normalised to the rpl13a gene. The normalised number of atrogin-1 (fbxo32) molecules in the samples obtained from the 5 µm LOV treatment group was considered to be 1 arbitrary unit (A.U.).

Niedbalska-Tarnowska J., Ochenkowska K., Migocka-Patrzałek M, & Dubińska-Magiera M. (2022). Assessment of the Preventive Effect of L-carnitine on Post-statin Muscle Damage in a Zebrafish Model. Cells, 11(8), 1297.

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Quantification of Gene Expression by RT-qPCR

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Total RNA was extracted using the Extracol reagent (EURx, Poland) according to the manufacturer’s guidelines. Using ImProm-II™ reverse transcriptase (Promega, Madison, WI, United States), 10 mg of total RNA was reverse-transcribed into cDNA. After diluting each cDNA sample with DEPC water to a total volume of 140 μL, 2 μL was used for real-time quantitative PCR (GoTaq^® qPCR Master Mix, Promega). Each cellular variant was measured in triplicate using the LightCycler^® 480 (Roche Applied Science) with initial denaturation at 95°C for 2 min, followed by 45 cycles of 95°C for 30 s and annealing at 60°C for 30 s. Primer sequences for references and genes of interest are presented in Supplementary Table S1. The amplification of specific transcripts was confirmed by the melting curves at the end of each PCR. The relative expression level was determined using appropriate references: H3F3A, RPLP0, and RPS17 were used for protein-encoding genes (UPF1, RBM22, and ZC3H12A), whereas U6 served as an endogenous control for the expression of selected ncRNAs (LINC01137, MIR6732, and MIR186). The Universal Human Reference RNA (Stratagene, La Jolla, CA, United States) was applied as a calibrator. LinRegPCR (v2021.2) was implemented to determine the baseline and measurements of real-time qPCR efficiency. The relative expression level was calculated using the Pfaffl method (Pfaffl, 2001 (link)).

Kołat D., Kałuzińska-Kołat Ż., Kośla K., Orzechowska M., Płuciennik E, & Bednarek A.K. (2023). LINC01137/miR-186-5p/WWOX: a novel axis identified from WWOX-related RNA interactome in bladder cancer. Frontiers in Genetics, 14, 1214968.

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