Dermal fibroblasts were obtained from patient 8 (3781-3781) by skin punch biopsy and cultured in amnioMAX C-100 complete medium (Life Technologies) as described previously12 (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) and treated with DNAseI to eliminate genomic DNA, according to manufacturer’s instructions. Complementary DNA (cDNA) synthesis was carried out using random oligomer primers and AMV Reverse Transcriptase (Roche). The cDNA samples were resolved on an E-Gel® electrophoresis system and extracted according to manufacturer’s instructions (ThermoFisher Scientific) in order 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.
Protocol detail
Fibroblast-based PUF60 Allele Sequencing
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Alleles
Biopsy
Cdna
Cell lines
Cultured medium
Electrophoresis
Fibroblast
Genomic
Patient
Primers
Reverse transcriptase
Skin
Synthesis
Corresponding Organization :
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
Top 5 similar protocols
Variable analysis
independent variables
- Skin punch biopsy from patient 8 (3781-3781)
- Expression levels of normal and mutant alleles of the PUF60 gene
- Primary skin-derived fibroblast cell lines from two sex-matched controls
- Positive control: Cell lines from two sex-matched controls
- Negative control: Not explicitly mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!