Fibroblast Transcriptome Analysis of Genetic Disorder
Dermal fibroblasts were obtained from patient 8 (3781–3781) by skin punch biopsy and cultured in amnioMAX C-100 complete medium (Life Technologies, Carlsbad, CA, USA) as described previously.12 (link) RNA was extracted from primary skin-derived fibroblast cell lines from patient 8 (3781–3781) and two sex-matched controls using RNeasy Kit (Qiagen, Hilden, Germany) and treated with DNAseI to eliminate genomic DNA, according to the manufacturer's instructions. Complementary DNA (cDNA) synthesis was carried out using random oligomer primers and AMV Reverse Transcriptase (Roche, Penzburg, Germany). The cDNA samples were resolved on an E-Gel electrophoresis system and extracted according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA) to sequence the amplicons corresponding to the normal and mutant alleles. Sequences of primers used for cDNA amplification and Sanger sequencing of PUF60 are available upon request.
Low K.J., Ansari M., Abou Jamra R., Clarke A., El Chehadeh S., FitzPatrick D.R., Greenslade M., Henderson A., Hurst J., Keller K., Kuentz P., Prescott T., Roessler F., Selmer K.K., Schneider M.C., Stewart F., Tatton-Brown K., Thevenon J., Vigeland M.D., Vogt J., Willems M., Zonana J., Study D.D, & Smithson S.F. (2017). PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features. European Journal of Human Genetics, 25(5), 552-559.
Other organizations :
St Michaels Hospital, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, University Hospital Leipzig, University Hospital of Wales, Hôpital d'Hautepierre, Southmead Hospital, Newcastle upon Tyne Hospital, Great Ormond Street Hospital, University College London, Oregon Health & Science University, Oslo University Hospital, Urbana University, University of Ulster, Belfast City Hospital, St George's Hospital, Wessex Regional Genetics Laboratory, Hôpital Arnaud de Villeneuve, Wellcome Sanger Institute, University of Bristol
Use of RNeasy Kit (Qiagen) and DNAseI treatment to extract and purify RNA
Use of random oligomer primers and AMV Reverse Transcriptase (Roche) for cDNA synthesis
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