The unigenes annotated as Avian Beta Defensin 2 (CL60147Contig), Avian Beta Defensin 13 (CL64604Contig1) and Cathelicidin 2 (CL31589Contig1) were selected for expression analysis. The expression profiles of the selected genes in 14 tissues of 3 crows were analyzed by real-time PCR using Roche -Light Cycler® 480 System. About 1 µg of total RNA extracted from each tissue was reverse transcribed using Superscript™ III First-Strand System for RT-PCR (Invitrogen). Real-time PCR was performed in 10 µl reaction containing cDNA sample, SYBR green mix, gene-specific primers (Table 4), and double-distilled water. The expression level of the three genes were measured by the 2(−ΔΔCt) method using β-actin as internal reference gene and oesophagus as calibrator tissue56 (link),120 (link)–122 (link).

Primer sequences for real time PCR.

Gene nameForward primerReverse primer
Avian beta-defensin 2 (AvBD-2)ACAGCCATGAAGATCCTTTACCGGCAAAGACAAACCTGGAGA
Avian beta-defensin 13 (AvBD-13)CAGCAGTGCAGAAGCAACCATTGCTGCAGCTCCCTTC
Cathelicidin 2 (CATH-2)CCGTGGATTCCTACAACCAGTCCATCATGCTGAAGTTGAGTC
β- actinCCCCACCTGAGCGTAAATACTCCTGCTTGCTGATCCACAT
The statistical analysis of the data was carried out using multiple t-test analysis and one-way ANOVA. Data were repeated in triplicate and plotted with GraphPad Prism 10. Differences were defined as significant if P < 0.05.
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