Genotyping melanocyte cells for rs398206

Melanocyte cells were grown in Dermal Cell Basal Medium (ATCC PCS-200-030) supplemented with Melanocyte Growth Kit (ATCC PCS-200-041) and 1% amphotericin B/penicillin/streptomycin (120-096-711, Quality Biological) as described before^{24 (link)}. Total RNA was isolated using a miRNeasy Mini kit (217004, Qiagen) further treated with CTAB-Urea following a previously described method^{68 (link)} to remove excess melanin pigmentation. cDNA was synthesized from total RNA using iScript Advanced cDNA Synthesis Kit (Bio-Rad). Genomic DNA and cDNA were then genotyped for rs398206 using custom Taqman genotyping probe set (ANRWEYM) recognizing both genomic DNA and cDNA (ENST00000543692) with a 5 bp 5’ overhang on the left primer for cDNA based on Ensembl archive 75 annotation. From a total of 44 samples heterozygous for rs398206, 27 samples passing QC (Ct values lower than 38 for both alleles in cDNA and genomic DNA) were used to calculate A/C allelic ratio based on dRn values.

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Choi J., Zhang T., Vu A., Ablain J., Makowski M.M., Colli L.M., Xu M., Hennessey R.C., Yin J., Rothschild H., Gräwe C., Kovacs M.A., Funderburk K.M., Brossard M., Taylor J., Pasaniuc B., Chari R., Chanock S.J., Hoggart C.J., Demenais F., Barrett J.H., Law M.H., Iles M.M., Yu K., Vermeulen M., Zon L.I, & Brown K.M. (2020). Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma. Nature Communications, 11, 2718.

Publication 2020

Melanocyte Growth Kit amphotericin B/penicillin/streptomycin miRNeasy Mini kit iScript Advanced cDNA Synthesis Kit

Allelic Amphotericin b Biological Cdna Cell Ctab Genomic Heterozygous Melanin Melanocyte Penicillin Pigmentation Primer Streptomycin Synthesis Urea

Corresponding Organization :

Other organizations : National Cancer Institute, Dana-Farber Cancer Institute, Boston Children's Hospital, Oncode Institute, Radboud University Nijmegen, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Université Paris Cité, Inserm, University of Leeds, University of California, Los Angeles, Frederick National Laboratory for Cancer Research, Imperial College London, QIMR Berghofer Medical Research Institute

Top 5 similar protocols

Variable analysis

independent variables

Genotype for rs398206

dependent variables

A/C allelic ratio

control variables

Melanocyte cells grown in Dermal Cell Basal Medium (ATCC PCS-200-030) supplemented with Melanocyte Growth Kit (ATCC PCS-200-041) and 1% amphotericin B/penicillin/streptomycin (120-096-711, Quality Biological)
Total RNA isolated using a miRNeasy Mini kit (217004, Qiagen) and further treated with CTAB-Urea to remove excess melanin pigmentation
CDNA synthesized from total RNA using iScript Advanced cDNA Synthesis Kit (Bio-Rad)
Ct values lower than 38 for both alleles in cDNA and genomic DNA

Annotations

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