98-P

• Published in: Human immunology Vol. 74; p. 119
• Main Authors: Yuan, Yao, Adams, Sharon D, Dinauer, David M, Rosenau, Christopher M, Uribe, Marcela R, Flegel, Willy A
• Format: Journal Article
• Language: English
• Published: 01.11.2013
• Subjects: Allergy and Immunology
• ISSN: 0198-8859
• DOI: 10.1016/j.humimm.2013.08.173

Aim To develop assays to quantify the mRNA transcribed from KIR genes utilizing a quantitative real-time PCR (qPCR) method. To study the KIR mRNA expression patterns in PBMC and enriched NK cells among healthy volunteers. Methods PBMCs were obtained from freshly drawn whole blood (maximum 48 hours) from 14 healthy volunteers using a Ficoll method. NK cell enrichment from PBMC was performed by negative selection using a magnetic bead kit. mRNA extraction and cDNA synthesis was followed by EvaGreen based qPCR. Relative KIR mRNA expression was quantified by 2-ΔΔCt algorithm with GAPDH as reference. qPCR with a Cq ⩽ 38.0 was considered positive. Results 10 mRNA expression assays for the 9 KIR genes 2DL1, 2DL5A, 2DL5B, 2DS1, 2DS2, 2DS3, 2DS4 (2DS4-full length and 2DS4-truncated), 2DS5 and 3DS1 have been developed and validated. Among the 14 individuals, KIR mRNAs were detected in all samples carrying respective KIR genes and were negative for KIR gene negative samples. The mRNA expression varied among individuals. In PBMC, the relative quantification (RQ) of cDNA showed the largest variation for 2DL1 (14-fold, n = 14) and the smallest for 3DS1 (1.8-fold, n = 3) among volunteers who were positive for the respective genes. 2DL5B cDNA was not detected, as expected for this gene. More KIR cDNA was detected in the enriched NK cell fraction when compared to PBMC, with the largest difference observed for 2DL1 (NK : PBMC ratio, mean ± SD: 37.8 ± 25.0, n = 14). Conclusions Development of qPCR assays with high specificity and easy applicability will be useful, because all known 15 KIR genes share > 90% identity at the genomic level. We established 10 assays for the mRNA expression of 9 KIR genes. KIR mRNA expression varied among healthy individuals carrying the same KIR genotype. Because NK cells are the predominant, but not the only source of KIR mRNA, analyzing PBMC and NK cells will allow establishing an overall and NK cell specific KIR mRNA expression signature from an individual.