Genetic Analysis of Patient Samples

After obtaining patients’ informed consent, genetic analyses and skin biopsies were performed. Genomic DNA was extracted from the peripheral blood of patients of interest using the MagNA Pure Compact System (Roche Diagnostics, Monza, Italy). Targeted mutation analysis was performed by Sanger sequencing. Sequencing was performed in an ABI 3500 genetic analyzer (Applied Biosystems, Foster City, CA, USA) using ABI BigDye 3.1 chemistry, and traces were analyzed with SeqScape software (https://www3.appliedbiosystems.com accessed on 26 January 2023). Total RNA was isolated from skin fibroblasts obtained from punch biopsies in P1 and P2. This was performed using a High Pure RNA Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany), and the cDNA products were used directly to co-amplify the housekeeping GAPDH and REEP1 transcripts.

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Naef V., Meschini M.C., Tessa A., Morani F., Corsinovi D., Ogi A., Marchese M., Ori M., Santorelli F.M, & Doccini S. (2023). Converging Role for REEP1/SPG31 in Oxidative Stress. International Journal of Molecular Sciences, 24(4), 3527.

Publication 2023

MagNA Pure Compact System ABI 3500 genetic analyzer High Pure RNA Isolation Kit

Abi 1 Biopsies Blood Cdna Diagnostics Fibroblasts Gapdh Genetic Genomic Isolation Patients Skin Targeted mutation

Corresponding Organization : Fondazione Stella Maris

Other organizations : University of Pisa

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Variable analysis

independent variables

Genetic analyses
Skin biopsies

dependent variables

Presence or absence of targeted mutations
Expression levels of GAPDH and REEP1 transcripts

control variables

Informed consent of patients
Use of MagNA Pure Compact System for DNA extraction
Use of Sanger sequencing for mutation analysis
Use of ABI 3500 genetic analyzer and BigDye 3.1 chemistry for sequencing
Use of High Pure RNA Isolation Kit for RNA extraction from skin fibroblasts
Use of GAPDH as a housekeeping gene for co-amplification with REEP1

controls

Positive control: Not specified
Negative control: Not specified

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