Computational protocol: Endothelial Cell Processing and Alternatively Spliced Transcripts of Factor VIII: Potential Implications for Coagulation Cascades and Pulmonary Hypertension

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Protocol publication

[…] RNA was extracted from EC at various degrees of confluency using Tri® Reagent (Sigma). Following quantification by optical densitometry, 1 µg of RNA was used for cDNA synthesis using Superscript™ II (Invitrogen, Paisley, UK). Oligonucleotide primers were designed to constitutive and alternative exons of the known FVIII transcripts (RefSeq variants 1; NM_000132 and 2; NM_019863), and to novel exons identified by the ExonMine project through extensive mining of BLAT alignments , , to human genome NCBI Build 36.1 of mRNA and expressed sequence tag (EST) sequences deposited in the GenBank . Rt-PCR products were purified by gel excision or treated with an ExoSAP mix comprising 1.2 u of ExoI and 1.06 u of SAP (Promega, Southampton, UK), sequenced on an ABI 3730x1 DNA analyser, and results analysed using FinchTV V1 4.0 software (Geospiza, Seattle, USA). Confirmation of FVIII transcript identity was obtained by Blastn ( Evolutionary conservation of flanking sequences was performed via the UCSC Genome Browser . […]

Pipeline specifications

Software tools BLAT, FinchTV, BLASTN
Databases UCSC Genome Browser
Applications Sanger sequencing, Genome data visualization
Organisms Homo sapiens
Diseases Hemophilia A, Hypertension, Hypertension, Pulmonary, Thrombosis, von Willebrand Diseases, Venous Thromboembolism