Utility of the Housekeeping Genes 18S rRNA, β‐Actin and Glyceraldehyde‐3‐Phosphate‐Dehydrogenase for Normalization in Real‐Time Quantitative Reverse Transcriptase‐Polymerase Chain Reaction Analysis of Gene Expression in Human T Lymphocytes

Scandinavian Journal of Immunology - Tập 59 Số 6 - Trang 566-573 - 2004
Á Bas1,2, Göte Forsberg1,3,2, Sten Hammarström1, Marie‐Louise Hammarström1
1Department of Clinical Microbiology, Immunology
2The first two authors contributed equally to this work.
3Department of Clinical Sciences, Pediatrics, Umea University, Umea, Sweden

Tóm tắt

AbstractThe accuracy of 18S rRNA, β‐actin mRNA and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real‐time reverse transcriptase‐polymerase chain reaction was used to determine the expression level of 18S rRNA, β‐actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T‐cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of β‐actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated γδTCR+, CD4+ and CD8+ subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30–70‐fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.

Từ khóa


Tài liệu tham khảo

10.2144/00292rv02

10.1677/jme.0.0290023

10.4049/jimmunol.171.7.3359

10.1046/j.1365-2249.2003.02035.x

10.1046/j.1365-2249.2003.02268.x

10.1093/nar/13.3.711

10.1097/00006676-199007000-00008

10.1016/0016-5085(91)90263-K

10.1111/j.1365-2184.1993.tb00029.x

10.1016/0531-5565(93)90032-9

Hodin RA, 1995, Pattern of rat intestinal brush‐border enzyme gene expression changes with epithelial growth state, Am J Physiol, 269, C385, 10.1152/ajpcell.1995.269.2.C385

10.1006/bbrc.1997.6198

10.1080/10495399809525893

Tokunaga K, 1987, Enhanced expression of a glyceraldehyde‐3‐phosphate dehydrogenase gene in human lung cancers, Cancer Res, 47, 5616

Schek N, 1988, Increased glyceraldehyde‐3‐phosphate dehydrogenase gene expression in human pancreatic adenocarcinoma, Cancer Res, 48, 6354

10.1006/bbrc.1999.0815

10.1006/mcpr.2000.0288

10.1152/physiolgenomics.00040.2001

10.1016/S0003-2697(02)00311-1

10.1006/abio.2001.5171

10.1186/gb-2002-3-7-research0034

10.1016/S0003-2697(02)00045-3

10.1016/0022-1759(92)90147-L

10.1136/gut.47.2.215

Lundqvist C, 1996, Intraepithelial lymphocytes in human gut have lytic potential and a cytokine profile that suggest T helper 1 and cytotoxic functions, J Immunol, 157, 1926, 10.4049/jimmunol.157.5.1926

10.4049/jimmunol.153.5.2302

10.1093/clinchem/45.2.297

10.1053/gast.2002.35355

10.1046/j.1365-2567.2002.01461.x

10.1016/S0165-022X(00)00129-9

10.1006/mcpr.2001.0392

10.1006/abio.2001.5369

10.1046/j.1365-3083.1999.00549.x

10.1016/S0022-1759(01)00489-6