Computational protocol: A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA

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Protocol publication

[…] Human Splicing Finder (, Marseille, France) and NetGene2 (, Lyngby, Denmark) softwares were used to in silico evaluate the possible effects of the identified variant on gene splicing. Splice site scores were evaluated with the ‘Splice Site Prediction by Neural Network Site’ software (, Berkeley, CA, USA). [...] Total RNA was isolated using the TRIzol® protocol (Life Technologies, Carlsbad, CA, USA) and retro-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The cDNAs were amplified using primer pairs specifically designed as follows: (i) a primer pair spanning BRCA1 exons 21 and 22 to include the region containing the splicing variant; in particular, the forward primer was designed on the 20/21 exon junction and the reverse primer annealed on exon 23, giving an amplification of 825 bp; and (ii) a primer pair amplifying 165 bp, with the forward primer complementary to 20/21 exon junction and the reverse primer annealing to 22/23 exon junction of the BRCA1 gene (). The specificity of both primer pairs was verified by using Primer Blast ( PCR reactions were carried out using the 5 PRIME MasterMix (Eppendorf, Hamburg, Germany), with the following conditions: 94 °C for 2 min, 94 °C for 40 s, 60 °C (for the first pair) and 62 °C (for the second pair) for 40 s and 65 °C for 40 s for 40 cycles, 65 °C for 10 min. Agarose gels, the DNA Chip 1 K (BioRad, Hercules, CA, USA) and the High Sensitivity D1000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA) were used to verify the amplification products. Next, Sanger sequencing was performed with an ABI 3130 capillary sequencer (Applied Biosystems Inc., Foster City, CA, USA). Sequences analysis was carried out with the SeqMan (DNASTAR, Inc., Madison, WI, USA) and CodonCode Aligner V3.5.7 (CodonCode Co., Centerville, MA, USA) tools. […]

Pipeline specifications

Software tools HSF, NetGene2, Primer-BLAST, CodonCode Aligner
Applications WGS analysis, qPCR
Organisms Homo sapiens
Diseases Breast Neoplasms, Hereditary Breast and Ovarian Cancer Syndrome