Computational protocol: The Turkey Ig-Like Receptor Family: Identification, Expression and Function

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Protocol publication

[…] For the identification of CHIR homologues in other bird species via databases, known CHIR sequences were used. Both NCBI blastx (http://blast.ncbi.nlm.nih.gov/Blast.cgi) browsing protein database using a translated nucleotide query and ensemble genome browser blat searches (http://www.ensembl.org/Multi/blastview) looking for CHIR homologues especially in turkey (Meleagris gallopavo, assembly Turkey_2.01) and zebra finch (Taeniopygio guttata, assembly taeGut3.2.4) were applied. For duck only data from Ensemble Pre were available (http://pre.ensembl.org/Anas_platyrhynchos/Info/Index). Furthermore the keyword search function of ensemble genome browser was used as well as EST databases. [...] A part of a turkey spleen was immediately taken in Ambion RNAlater (Invitrogen life technologies, Darmstadt, Germany) and after storage over night at 4°C RNA was isolated by Trizol reagent (Peqlab, Erlangen, Germany). PBMC of a blood sample (Bxx line) were prepared by density centrifugation using Ficoll (Biochrom, Berlin, Germany) and subsequently RNA was isolated. Both spleen and PBMC RNA were reverse transcribed using the ThermoScipt™ RT-PCR System (Invitrogen life technologies, Darmstadt, Germany). Primers were designed according to partial sequences in the database and synthesized (Eurofins MWG Operon, Ebersberg, Germany). 5′ATGGCACCAATGGCGCTGGC-3′ was used as a common sense primer for amplifying TILR. 5′-CCTGCAGGGCTCCCATCTCT-3′ served as anti-sense primer for TILR-A, 5′-CACGGTAATTCAGTGCTCACTGTGG-3′ for TILR-AB and 5′-CGTGCCCACCTCGGCGTAGATA-3′ for TILR-B. Using Herculase II Fusion DNA-Polymerase (Agilent technologies, Waldbronn, Germany) PCR conditions were as follows: initial denaturation for 1 min at 94°C, three cycles at 94°C for 20 s, 65°C for 20 s and 68°C for 1 min. After that every three cycles the annealing temperature was decreased in steps of 1.0°C as far as the 21st cycle was finished. Then 13 cycles at 94°C for 20 s, 58°C for 20 s and 68°C for 1 min were added followed by a final extension at 68°C for 4 min. After purification with Wizard®SV Gel and PCR Clean-up system (Promega, Mannheim, Germany) the PCR product was cloned into pcR® Blunt II TOPO® vector (Invitrogen life technologies, Darmstadt, Germany). Colonies were tested by PCR and the plasmids from positive colonies were isolated using PureYield™ Plasmid Miniprep system (Promega, Mannheim, Germany) and sequenced (GATC, Konstanz, Germany). Sequence analysis and alignment were performed using NCBI database and DNASTAR Lasergene software package (Madison, USA). All new data has been deposited in GenBank, accession numbers are indicated in the figure legends. […]

Pipeline specifications

Software tools BLASTX, BLAT, DNASTAR Lasergene
Applications Sanger sequencing, Genome data visualization
Organisms Gallus gallus, Meleagris gallopavo, Anas platyrhynchos, Taeniopygia guttata
Diseases Chickenpox