Authentifiler pcr amplification kit

Manufactured by Thermo Fisher Scientific

Sourced in France, United States

The AuthentiFiler PCR Amplification Kit is a laboratory tool designed for DNA amplification. It enables the production of multiple copies of specific DNA sequences through the process of Polymerase Chain Reaction (PCR).

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11 protocols using authentifiler pcr amplification kit

Variant Pathogenicity Assessment Protocol

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Only nonsynonymous, splice-site and frameshift changes were considered for pathogenicity assessments (Table 2). Variants were classified according to the interpretation guidelines from the American
College of Medical Genetics and Genomics–Association for Molecular Pathology (ACMG–AMP).[28 (link)] Briefly, a variant was classified as likely/pathogenic if it arose de novo (or
from a somatic mosaic parent) and was not found in the publically available control datasets (ExAC v0.31, ESP6500, 5000 Genomes,
gnomAD r2.0.2).[21 –23 (link)] In cases where DNA
from both parents was unavailable for segregation analysis, likely/pathogenicity was inferred on the basis of (1) the variant type
(truncations and splice variants were seen as likely pathogenic), (2) recurrence (previously recorded as disease-causing in the
literature or disease databases (3) analysis with in silico pathogenicity prediction tools (CADD, PolyPhen-2, and
GERP), where all outputs had to be in agreement (CADD > 25, PolyPhen-2 > 0.9, and GERP > 5). Microsatellite
analysis (Authentifiler™ PCR Amplification kit, ThermoFisher Scientific) was performed on of all parents of probands with a
de novo variants to confirm parentage.

Esterhuizen A.I., Mefford H.C., Ramesar R.S., Wang S., Carvill G.L, & Wilmshurst J.M. (2018). DRAVET SYNDROME IN SOUTH AFRICAN INFANTS: TOOLS FOR AN EARLY DIAGNOSIS. Seizure, 62, 99-105.

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Cell Culture Protocols for Various Cell Lines

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H1299, HCT116, IMR90, 293T and HAFF cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum. P493-6 and MCF7 cell lines were cultured in RPMI medium 1640 containing 10% fetal bovine serum. MCF10A cell line were cultured in DMEM/F12 medium containing 5% horse serum, 20 μg/mL EGF, 0.5 μg/mL hydrocortisone, 100 ng/mL Cholera Toxin and 10 μg/mL insulin. P493-6 cells carrying a c-Myc tet-off system were provided by professor Ping Gao. All other cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were tested by STR profiling (GenePrint 10 System kit from Promega and AuthentiFiler PCR Amplification Kit from ThermoFisher) to authenticate the identity. All cells were tested for mycoplasma contamination by Cell Culture Contamination Detection Kit (ThermoFisher).

Cao L., Zhang P., Li J, & Wu M. (2017). LAST, a c-Myc-inducible long noncoding RNA, cooperates with CNBP to promote CCND1 mRNA stability in human cells. eLife, 6, e30433.

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STR profiling of patient tumors and PDX

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Patient tumors and corresponding PDX DNA samples were subjected to STR using the Authentifiler PCR amplification kit (Thermo Fisher Scientific, Illkirch, France) that amplifies nine unique STR loci and the amelogenin gender-determining marker, according to the manufacturer’s instructions. PCR products were separated by capillary electrophoresis on ABI PRISM 3100, and results were analyzed using the GeneMapper software.

Lang H., Béraud C., Cabel L., Fontugne J., Lassalle M., Krucker C., Dufour F., Groeneveld C.S., Dixon V., Meng X., Kamoun A., Chapeaublanc E., De Reynies A., Gamé X., Rischmann P., Bieche I., Masliah-Planchon J., Beaurepere R., Allory Y., Lindner V., Misseri Y., Radvanyi F., Lluel P., Bernard-Pierrot I, & Massfelder T. (2022). Integrated molecular and pharmacological characterization of patient-derived xenografts from bladder and ureteral cancers identifies new potential therapies. Frontiers in Oncology, 12, 930731.

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Sanger Sequencing and Phasing of LZTR1 Variants

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PCR amplification of LZTR1 was performed as described in Table S1. An additional amplicon was included to capture intron 16 where variants can lead to retention of an alternative exon.15 Following enzymatic purification, Sanger sequencing was performed using BigDye (version 3.1) and the ABI 3730XL (Applied Biosystems, Foster City, California).
Where parental samples were unavailable, compound‐heterozygous variants were phased by allele‐specific PCR whereby the 3′‐base of the first primer was complementary to either the wild‐type or mutant allele (Table S1). Sanger sequencing was then performed to determine the sequence at the second locus. A similar method was used to phase a de novo variant where the closest informative SNP was too distant to be phased by Illumina read‐pairs (Table S1).
For one family where the patient underwent exome sequencing as a singleton, maternity/paternity were confirmed by genotyping nine short tandem repeat (STR) loci using the AuthentiFiler PCR Amplification Kit (ThermoFisher Scientific, Waltham, Massachusetts).

Pagnamenta A.T., Kaisaki P.J., Bennett F., Burkitt‐Wright E., Martin H.C., Ferla M.P., Taylor J.M., Gompertz L., Lahiri N., Tatton‐Brown K., Newbury‐Ecob R., Henderson A., Joss S., Weber A., Carmichael J., Turnpenny P.D., McKee S., Forzano F., Ashraf T., Bradbury K., Shears D., Kini U., de Burca A., Blair E., Taylor J.C, & Stewart H. (2019). Delineation of dominant and recessive forms of LZTR1‐associated Noonan syndrome. Clinical Genetics, 95(6), 693-703.

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Short Tandem Repeat Analysis Protocol

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Short tandem repeat analysis was carried out using an AuthentiFiler PCR Amplification Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

Sato T., Muramatsu T., Tanabe M, & Inazawa J. (2018). Identification and characterization of transforming growth factor beta‐induced in circulating tumor cell subline from pancreatic cancer cell line. Cancer Science, 109(11), 3623-3633.

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Cell Culture Conditions for HCC Research

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Human HCC cell MHCC97L was a gift from Z. Y. Tang of Fudan University (Shanghai, China). CLC6 and CLC11 are gifts from L. Hui of Chinese Academy of Sciences (Shanghai, China). Human HCC cell Hep3B and mouse HCC cell Hepa1–6 were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines within this study were authenticated with the AuthentiFiler PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA). Cell cultures were routinely tested for mycoplasma infection. MHCC97L, Hep3B, and Hepa1–6 were cultured in Dulbecco’s modified Eagle’s medium (DMEM)–high glucose media with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) supplementations (all reagents are from Gibco by Life Technologies, Grand Island, NY, USA). CLC6 and CLC11 were cultured in RPMI 1640 medium (Gibco) with 10% FBS and 1% PS supplemented with epidermal growth factor (40 ng/ml; PeproTech, Cranbury, NJ, USA) and 1% Insulin-Transferrin-Selenium (Gibco). For in vitro culture of immune cell assays, cells were cultured in RPMI 1640 media with 10% FBS and 1% PS supplemented with 25 mM Hepes buffer (Gibco) and 50 μM 2-mercaptoethanol (Sigma-Aldrich). Cells were cultured in a 37°C humidified incubator supplied with 5% CO₂. Hypoxic environment was created by culturing HCC cells in 1% O₂ in a hypoxia incubator chamber for 24 or 48 hours as indicated.

Cheu J.W., Chiu D.K., Kwan K.K., Yang C., Yuen V.W., Goh C.C., Chui N.N., Shen W., Law C.T., Li Q., Zhang M.S., Bao M.H., Wong B.P., Chan C.Y., Liu C.X., Sit G.F., Ooi Z.Y., Deng H., Tse A.P., Ng I.O, & Wong C.C. (2023). Hypoxia-inducible factor orchestrates adenosine metabolism to promote liver cancer development. Science Advances, 9(18), eade5111.

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Neuroblastoma Cell Line Characterization

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MYCN amplified human neuroblastoma cell lines, NBLW and NBLW-R were cultured in RPMI 1640 (Sigma, Dorset, England) supplemented with 10% v/v Foetal Calf Serum (Gibco/Life Technologies Ltd, Paisley, UK). Photomicrographs (×20) were captured using a VisiCam® digital camera and analyser software (VWR International Ltd, Lutterworth, UK). DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Manchester, UK) according to the manufacturer's instructions, and quantified using the Nanodrop (Thermo Scientific, Waltham, MA USA). Cell line authentication was conducted using the AuthentiFiler™ PCR Amplification Kit (Applied Biosystems/Life Technologies Ltd) and Promega PowerPlex® 16 HS System according to manufacturer's protocols.

Chen L., Humphreys A., Turnbull L., Bellini A., Schleiermacher G., Salwen H., Cohn S.L., Bown N, & Tweddle D.A. (2016). Identification of different ALK mutations in a pair of neuroblastoma cell lines established at diagnosis and relapse. Oncotarget, 7(52), 87301-87311.

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Authenticated Human and Mouse HCC Cell Lines

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Multiple human HCC cell lines including MHCC97L, PLC/PRF/5, Huh7, and a mouse HCC cell line (Hepa 1-6) were used in this study. MHCC97L and Huh7 were kind gifts from Dr Z.Y. Yang of Fudan University (Shanghai, China) and Professor H. Nakabayashi of Hokkaido University School of Medicine (Sapporo, Japan), respectively. PLC/PRF/5 and Hepa 1-6 were obtained from American Type Culture Collect (Manassas, VA). All cell lines were cultured in Dulbecco’s Modified Eagle Medium-high glucose media with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) supplementations (all reagents from Gibco by Life Technologies, Grand Island, NY). Cells were cultured in a 37 °C humidified incubator supplied with 5% CO₂. Authentication of all cell lines within this study was performed using the AuthentiFiler PCR Amplification Kit (Applied Biosystems, Foster City, CA). All authenticated cell stocks were thawed and used within 4 passages.

Cheu J.W., Lee D., Li Q., Goh C.C., Bao M.H., Yuen V.W., Zhang M.S., Yang C., Chan C.Y., Tse A.P., Sit G.F., Liu C.X., Ng I.O., Wong C.M, & Wong C.C. (2023). Ferroptosis Suppressor Protein 1 Inhibition Promotes Tumor Ferroptosis and Anti-tumor Immune Responses in Liver Cancer. Cellular and Molecular Gastroenterology and Hepatology, 16(1), 133-159.

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Genetic Typing of Mosquito Blood Meals

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Genetic typing of blood meal samples has been described in detail elsewhere (Gonçalves et al., 2017 (link)). Briefly, bloodfed mosquitoes’ abdomens were processed using Boom extraction method (Boom et al., 1990 (link)). The Authentifiler PCR Amplification kit (Applied Biosystems), with nine human microsatellite markers and one gender marker, was used to compare human DNA in blood meals and in blood samples collected from study participants. Capillary electrophoresis was used to determine DNA profiles. Mosquito blood meals with more than two alleles in at least three loci were considered to have multiple human sources.

Guelbéogo W.M., Gonçalves B.P., Grignard L., Bradley J., Serme S.S., Hellewell J., Lanke K., Zongo S., Sepúlveda N., Soulama I., Wangrawa D.W., Yakob L., Sagnon N., Bousema T, & Drakeley C. (2018). Variation in natural exposure to anopheles mosquitoes and its effects on malaria transmission. eLife, 7, e32625.

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STR Profiling of Tumor DNA

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DNA from patients' primary tumors and from corresponding tumors at passage ranging from P1 to P4 was obtained by phenol/chloroform extraction A nanodrop ND-1000 spectrophotometer (Thermo scientific, Illkirch, France) was used to determine DNA concentration and purity. DNA samples were subjected to short tandem repeat (STR) DNA fingerprinting using the AuthentiFiler PCR amplification Kit (Life technologies, Saint Aubin, France) that amplifies 9 unique STR loci (8 that comprise tetranucleotide repeat units and one locus trinucleotide) and the Amelogenin gender-determining marker, according to manufacturer instructions. PCR products were separated by capillary electrophoresis on a genetic analyzer ABI PRISM 3100 and results analyzed using the GeneMapper software.

Lang H., Béraud C., Bethry A., Danilin S., Lindner V., Coquard C., Rothhut S, & Massfelder T. (2016). Establishment of a large panel of patient-derived preclinical models of human renal cell carcinoma. Oncotarget, 7(37), 59336-59359.

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