Optimal reference genes for qPCR in resting and activated human NK cells--Flow cytometric data correspond to qPCR gene expression analysis.
Natural killer cells (NK cells) are cytotoxic lymphocytes critical to the innate immune system engaged in rapid response against tumor or virus infected cells. After activation NK cells acquire enhanced cytotoxicity and are capable of producing cytokines to stimulate other immune cells. Quantitative PCR (qPCR) is a method of choice for gene expression analysis but the usage of reliable reference genes for the normalization process is critical. Commonly used reference genes may vary in expression level between different experimental conditions providing wrong quantitative results of the studied genes' expression levels. Fourteen potential endogenous control genes were analyzed by qPCR method in NK-92 cell line that shows characteristics of human natural killer cells and is often used in studies on biology of NK lymphocytes. NK-92 cells were stimulated with IL-2 or TNF for 2, 24 or 72 h. Results were analyzed with RefFinder, a program which enables evaluation and screening of reference genes and integrates the currently available major computational programs (Genorm, Normfinder, BestKeeper and Delta Ct). The most stable gene in activated and non-activated NK cells was B2M, followed by IPO-8 and GAPDH and the least stable were HPRT1, PPIA and RPL32. The normalization process was performed on SOD2 gene and the results of qPCR experiments were confirmed by flow cytometry. The flow cytometric data corresponded to the results of qPCR gene expression analysis performed for the reference genes qualified by RefFinder as the most stable.
Department of Histology, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: lkras@gumed.edu.pl.
Lucyna
L
Kaszubowska
Department of Histology, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: pwierzb@gumed.edu.pl.
Piotr Mieczysław
PM
Wierzbicki
Department of Histology, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: sylwia.karsznia@gmail.com.
Sylwia
S
Karsznia
Department of Histology, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: damskama@gmail.com.
Marta
M
Damska
Department of Histology, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: t.slebioda@gumed.edu.pl.
Tomasz Jerzy
TJ
Ślebioda
Department of Social and Clinical Gerontology, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: jfoerster@gumed.edu.pl.
Jerzy
J
Foerster
Department of Histology, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: zkmiec@gumed.edu.pl.
Zbigniew
Z
Kmieć
D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology; D007694:Killer Cells, Natural / Q000166:cytology* / Q000276:immunology*; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012015:Reference Standards; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D001613:beta 2-Microglobulin / Q000235:genetics; D028961:beta Karyopherins / Q000235:genetics
D016207:Cytokines; C508594:IL2 protein, human; C107282:IPO8 protein, human; D007376:Interleukin-2; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D001613:beta 2-Microglobulin; D028961:beta Karyopherins; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)
HPRT1; NK cell activation; NK-92 cells; Reference genes; qPCR; β-2-microglobulin