Computational protocol: Conservation of CD44 exon v3 functional elements in mammals

Similar protocols

Protocol publication

[…] Frozen (-80°C) blood samples were selected from the animal tissue bank of the Department of R+D+I, Laboratorio Dr. Echevarne, Barcelona, Spain. Genomic DNA was isolated from 200 μl of blood using the NucleoSpin Blood kit (Macherey-Nagel) following manufacturer's instructions. PCR amplification of CD44v3 was performed with INT6SF and I7wtR primer set or -49v3F and I7wtR primer set (table ) using PCR Master Mix (Promega). PCR bands of interest were isolated from agarose using the NucleoSpin Extract II kit (Machery-Nagel), sequenced in both directions with the primers used during PCR and the CEQ Dye Terminator Cycle Sequencing Quick Start kit (Beckman Coulter) and analyzed in a CEQ 8800 Genetic Analysis System (Beckman Coulter). All sequences were edited to remove ambiguous base calls and primer sequences and submitted to GenBank.CD44 RT-PCR was performed from total RNA extracted from frozen blood samples with a modification of the QIAamp RNA Blood Mini kit protocol (QIAGEN). Briefly, 150 μl of frozen blood were lysed at 70°C for 10 min with RLT/β-mercaptoethanol buffer containing 4 mg/ml Proteinase K and centrifuged at 10,000 × g for 3 min. 450 μl of the lysate supernatant were mixed with 225 μl of absolute ethanol and loaded in a QIAamp spin column following manufacturer's instructions. Eluted RNA was treated with RQ1 RNase-free DNase (Promega) at 37°C for 30 min and purified following the QIAamp RNA Mini protocol for RNA cleanup (QIAGEN). The first-strand reaction was performed with random primers (Promega) and SuperScript II Reverse Transcriptase (Invitrogen). As control of RNA quality, total CD44 isoforms were amplified with degenerate E20F-VI and E20R-QEM primer set (table ) using GC-Rich PCR System (Roche).In order to amplify CD44v3 containing isoforms, PCR primers were designed based on a multiple sequence alignment containing the sequences corresponding to the 95 mammalian species. Exon v3 positions that showed full conservation were identified and selected to locate the 3' ends of the primers ensuring perfect matches. According to this, v3 amplification was perfomed with primers 13v3F and 100v3R (table ) and PCR Master Mix (Promega). As control of complete genomic DNA digestion, non reverse-transcribed RNAs were tested amplification negative with primers 13v3F and 100v3R.Molecular conservation analyses were conducted using the MEGA version 3.1 software [] and sequence logos were generated with the WebLogo application []. […]

Pipeline specifications

Software tools MEGA, WebLogo
Application Genome data visualization
Organisms Homo sapiens