The five breast cancer cell lines were genotyped upon receipt from ATCC and after expansion using the AmpFLSTR Identifiler Plus PCR Amplification Kit (Life Technologies Cat# 4427368) on a 3500xl Genetic Analyzer with a 36-cm capillary array and POP-4 polymer (Life Technologies). PCR amplification was carried out in a total 12.5 μL reaction volume (1/2 reactions) with 1 μL of purified genomic DNA (1.0 ng/μL) on GeneAmp PCR System 9700 Cycler (Life Technologies) for 28 cycles according to the conditions specified by the manufacturer. Fifteen short tandem repeat (STR) loci with Amelogenin (sex-typing marker) were co-amplified in a single tube. After the reaction, 0.5 μL of GeneScan 600 LIZ Size Standard v2.0 and 8.5 μL Hi-Di Formamide (Life Technologies) were added to 1 μL of the PCR product or allelic ladder for a total volume of 10 μL. The samples were analyzed on the 24-capillary 3500xl Genetic Analyzer without prior denaturation of samples. Samples were injected electrokinetically for 15 s at 1.2 kV. The STR alleles were then separated at 15 kV at a run temperature of 60 °C. Data from the 3500xl was analyzed using GeneMapper ID-X software (version 1.3; Life Technologies).
Genemapper id x software
Manufactured by Thermo Fisher Scientific
Sourced in United States
GeneMapper ID-X software is a powerful tool for DNA fragment analysis. It is designed to accurately size and identify DNA fragments, enabling researchers to perform genetic profiling and identification. The software provides a robust and efficient platform for analyzing data from various capillary electrophoresis instruments.
Automatically generated - may contain errors
Lab products found in correlation
Geneamp pcr system 9700, by Thermo Fisher Scientific (4 mentions)
Abi 3500 genetic analyzer, by Thermo Fisher Scientific (4 mentions)
3500xl genetic analyzer, by Thermo Fisher Scientific (3 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (3 mentions)
Hi di formamide, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 3500xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 3500 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Proflex 96 well pcr system, by Thermo Fisher Scientific (2 mentions)
Pop 4 polymer, by Thermo Fisher Scientific (1 mentions)
Powerplex 21 system, by Promega (1 mentions)
Dna iq system, by Promega (1 mentions)
3500xl genetic analyser, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Powerplex 16 hs, by Promega (1 mentions)
Abi3500 automated sequencer, by Thermo Fisher Scientific (1 mentions)
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Yfiler platinum pcr amplification kit, by Thermo Fisher Scientific (1 mentions)
Powerplex 18d kit, by Promega (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
28 protocols using genemapper id x software
1
Genotyping Breast Cancer Cell Lines
2
DNA Extraction and Analysis Protocol
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Sampling was conducted by applying a wet-dry swabbing technique using viscose swabs (Forensic Swab L, Sarstedt, Nümbrecht, Germany). Upon collection, wet and dry swabs were returned to their respective tube bases, which contained a ventilation membrane for self-drying, and placed into a labelled envelope for storage. Air-dried swab tips were excised and placed into tubes (wet/dry swabs constituted one sample) and stored at −20 °C until the commencement of DNA analysis. DNA was extracted using the DNA IQ system (Promega, Madison, WI, USA) to a final volume of 60 μL and quantified with Quantifiler® Trio DNA Quantifiler Kit on an ABIPRISM® 7500 (Life Technologies, Carlsbad, CA, USA) and interpreted using HID software. Amplification was carried out using the PowerPlex®21 system (Promega, Madison, WI, USA) for 30 cycles using 0.5 ng of template DNA, or, if the sample concentration was below 0.033 ng/μL,15 μL of the sample was used. Amplified product detection and sizing was performed on a 3500 xL Genetic Analyser (Life Technologies, Carlsbad, CA, USA) with an injection voltage of 1.2 kV and an injection time of 24 s. GeneMapper® ID-X software (v1.5, Life Technologies, Carlsbad, CA, USA) was used for genotyping, with an analytical threshold of 175 RFU, as per laboratory protocol.
3
Multiplex STR Loci Amplification
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To amplify the STR 13 regions (loci) (D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, D16S539, D7S820, D13S317, D5S818, VWA, D18S51, and CSF1PO), as well as amelogenin to identify gender, we used the AMPFℓSTR Identifiler ® Plus ® PCR Kit (Life Technologies, Carlsbad, CA, USA) and PowerPlex ® 16HS (Promega) commercial kits following the manufacturer protocols. Amplicon separation and detection were performed using the ABI3500 automated sequencer (Applied Biosystems; Foster City, CA, USA) with the GS-600 LIZ standard and POP 4 polymer (Applied Biosystems) size. Genotyping was performed using the GeneMapper IDX software (Life Technologies).
4
Quantifying Donor Cell Chimerism
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STR-PCR was used to assess DC. According to the manufacturer's protocol and the instructions of the ABIPRISM 3100 Genetic Analyzer (Thermo Fisher Scientific, Inc.), DC was detected in all patients receiving HSCT. Detection was performed at days 7, 14 and 21 after transplantation and in patients with recurrence, and the results were analysed using GeneMapper ID-X software (version 1.2; Thermo Fisher Scientific, Inc.). According to the effective site of peak area percentage, quantitative calculation of donor cells was performed using the following formula: DC ratio = donor area/(donor area + recipient area).
5
Multiplex Y-STR and Y-InDel Genotyping
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Thirty‐eight Y‐STR loci plus 3 Y‐InDels were co‐amplified using the Yfiler™ Platinum PCR Amplification Kit in a GeneAmp PCR 9700 thermal cycler (Thermo Fisher Scientific) following the manufacturer's instructions. Amplified fragments were detected by capillary electrophoresis on the Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific). The electrophoresis data were automatically analyzed by GeneMapper® ID‐X software (Thermo Fisher Scientific). Negative control (H2O) and positive control (007) were genotyped in each batch of DNA amplification. We strictly followed the recommendations for the DNA commission of the International Society of Forensic Genetics (ISFG) in the present study (Gusmao et al., 2006 ).
6
Microsatellite-based Pedigree Reconstruction
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At the end of the experiment, both parents and offspring were euthanized by overdose of buffered MS‐222 and individually stored in 70% ethanol at −5°C. DNA was extracted from tail tissue and processed according to the protocol described in (Becher et al., 2002 ). Fish were genotyped fish at six autosomal microsatellite loci (see Table S1 for details) as described in (Becher et al., 2002 ) and (Bergero et al., 2019 (link)). Polymerase chain reaction (PCR) reaction conditions and the molecular protocol are described in full in Supplementary Information S2 . Individual fish were genotyped using Genemapper® ID‐X software (Thermofisher Scientific, Waltham, MA, USA) by scoring genotypes across all six microsatellite markers. Pedigree reconstruction was enabled by the program COLONY 2.0.4.5 (http://www.zsl.org/science/software/COLONY ), which reconstructs parental genotypes from offspring genotypes using maximum likelihood (Jones & Wang, 2010 (link); Wang & Santure, 2009 (link)). The details of COLONY run parameters can be seen in Supplementary Information S3 .
7
Genetic Diversity Assessment of Sri Lankan Populations
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The study was conducted with the approval of the Ethics Review Committee, Institute of Biology, Sri Lanka (ERC IOBSL 135 11 15) and the study was performed in line with the principles of the Declaration of Helsinki. Written informed consent was obtained from all individual participants included in the study. Finger pricked blood samples were collected from 838 unrelated individuals from the four ethnic groups in the Sri Lankan population; 426 samples from Sinhalese (60.6% males), 154 samples from the Sri Lankan Tamils (50% males), 128 samples from Indian Tamils (49.2% males), and 130 samples from Sri Lankan Moors (51.5% males). Genomic DNA was extracted using Chelex-100 method60 (link) and subjected to PCR amplification using the single tube 16 X-STR multiplex system described in Perera et al.15 (link). Amplified products were resolved with capillary gel electrophoresis using ABI 3500 Genetic Analyzer (Thermo Fisher Scientific, USA) and data analysis allele designation was performed using GeneMapper IDX software (Thermo Fisher Scientific, USA).
8
Cell Line Authentication and Mycoplasma-Free Culture
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Breast cancer cell lines MDA‐MB‐231 (RRID: CVCL_0062), MCF‐7 (RRID: CVCL_0031) and BT‐549 (RRID: CVCL_1092) were obtained from American Type Culture Collection (ATCC, Manassas, VA). Hair‐follicle‐derived stem cell and dermal fibroblast were isolated as described previously [27 (link)]. All cells except BT‐549 were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 1% (v/v) antibiotic‐antimycotic cocktail (Thermo Fisher Scientific, Grand Island, NY, USA) and 10% (v/v) fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA). BT‐549 was cultured in RPMI (Thermo Fisher Scientific) supplemented with 1% (v/v) antibiotic‐antimycotic cocktail, 10% (v/v) fetal bovine serum and 0.023 U·mL−1 insulin. All cell lines have been authenticated in the past 3 years by STR profiling at ATCC. According to the vendor, the commercially available PowerPlex® 18D kit (Promega, Madison, WI, USA) was used to amplify 17 short tandem repeat (STR) loci plus the gender‐determining locus, Amelogenin. Samples were then processed with the ABI Prism® 3500xl Genetic Analyzer (Thermo Fisher Scientific), and data were analyzed using GeneMapper® ID‐X software (Thermo Fisher Scientific) to confirm cell identity and purity. Finally, all experiments were performed with mycoplasma‐free cells.
9
Multiplex PCR Amplification and STR Profiling
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About 1.0 mm2 of the bloodstain sample was directly amplified by the multiplex PCR system of 44 loci on GeneAmp PCR system 9,700 (Thermo Fisher Scientific), following the manufacturer's instructions. The 10 µl total volume of multiplex reaction was consist of the bloodstain sample, 5 µl of Master Mix, 2.5 µl of Primer Mix, and 2.5 µl nuclease‐free water. The thermal cycling parameter was set as 95°C for 5 min, 30 cycles of 94°C for 10 s, 60°C for 1 min, 72°C for 30 s, and then 60°C for 15 min and stored at 4°C. Subsequently, 1.0 µl PCR product was mixed with 0.5 µl internal standard SIZ‐580 (Health GeneTech) and 8.5 µl Hi‐Di deionized formamide for denaturation at 95°C for 3 min and cooled at 4°C for 3 min. Finally, Applied Biosystems® 3500 xL Genetic Analyzer (Thermo Fisher Scientific) was used for STRs and InDels genotyping. GeneMapper ID‐X software (Thermo Fisher Scientific) was used to analyze electrophoresis results.
10
Y-STR Profiling of Bone Powder
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Human genomic DNA was amplified with reagents from the AmpFlSTR® Yfiler™ PCR Amplification Kit and a GeneAmp® PCR System 9700 (Thermo Fisher Scientific), according to manufacturer’s recommendations [31 ]. Negative controls consisted of 10 μl low-TE buffer and 10 μl 9947A female DNA (0.1 ng/μl); 10 μl 007 Male Control DNA (0.1 ng/μl) served as the positive control. PCR products were separated via capillary electrophoresis (CE) on a 3500xl Genetic Analyzer, and analyzed using GeneMapper® ID-X software (Thermo Fisher Scientific). DNA (elution #1 and elution #2) from seven bone powder fractions was typed.
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