CFTR Gene Sequencing and Analysis

Genomic DNA was extracted from both the peripheral blood leucocytes of enrolled individuals and derived undifferentiated CF-CRC-AESC, using the QIAamp DNA Blood midi kit (Qiagen, Hilden, Germany) and quantified using fluorimetry (Qubit, Invitrogen, CA, USA). The proximal 5′-flanking, all exons and adjacent intronic zones, and the 3′-UTR of the CFTR gene (RefSeq NM_000492.4, NG_016465.4) were sequenced by a Sanger cycle sequencing protocol (ThermoFisher Scientific, Waltham, MA, USA) as previously described [21 (link),22 (link),23 (link)], using a genetic analyzer (ABI PRISM 3130xl; ThermoFisher Scientific). Genetic analysis was completed through multiplex ligation-dependent probe amplification (SALSA MLPA probemix P091 CFTR, MRC Holland, Amsterdam, The Netherlands) to unveil macrodeletions/macroduplications. The genotype of derived undifferentiated CF-CRC-AESC was confirmed using the same methodology. Results of mutational analysis have already been published [5 (link),19 (link)].

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Bruno S.M., Blaconà G., Lo Cicero S., Castelli G., Virgulti M., Testino G., Pierandrei S., Fuso A., Cimino G., Ferraguti G., Eramo A, & Lucarelli M. (2023). Quantitative Evaluation of CFTR Gene Expression: A Comparison between Relative Quantification by Real-Time PCR and Absolute Quantification by Droplet Digital PCR. Genes, 14(9), 1781.

Publication 2023

QIAamp DNA Blood midi kit Sanger cycle sequencing protocol ABI PRISM 3130xl SALSA MLPA probemix P091 CFTR

Blood Cftr Exons Fluorimetry Gene Genetic Genomic Genotype Intronic Leucocytes Mlpa Mutational Prism

Corresponding Organization : Sapienza University of Rome

Other organizations : Istituto Superiore di Sanità, Policlinico Umberto I

Top 5 similar protocols

Variable analysis

independent variables

Genomic DNA extracted from peripheral blood leucocytes of enrolled individuals
Genomic DNA extracted from derived undifferentiated CF-CRC-AESC

dependent variables

Sequencing of the proximal 5′-flanking, all exons and adjacent intronic zones, and the 3′-UTR of the CFTR gene
Genetic analysis through multiplex ligation-dependent probe amplification (MLPA) to unveil macrodeletions/macroduplications

control variables

QIAamp DNA Blood midi kit (Qiagen, Hilden, Germany) for DNA extraction
Fluorimetry (Qubit, Invitrogen, CA, USA) for DNA quantification
Sanger cycle sequencing protocol (ThermoFisher Scientific, Waltham, MA, USA) for gene sequencing
Genetic analyzer (ABI PRISM 3130xl; ThermoFisher Scientific) for genetic analysis

positive controls

Undifferentiated CF-CRC-AESC genotype confirmed using the same methodology

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