RT-PCR amplification of various canine cytokines and so-called house-keeping genes in a species-specific macrophage cell line (DH82) and canine peripheral blood leukocytes.
Total ribonucleic acid (RNA) isolated from a continuous canine macrophage cell line (DH82) was used in reverse transcription polymerase chain reactions (RT-PCR) for the detection of transcripts of interleukin (IL)-8, -12, and tumour necrosis factor-alpha (TNF). Three different methods of RNA isolation (standard guanidinium-thiocyanate method with and without application of RNA matrix, and boiling) were used and compared in regard to RT-PCR results. The most suitable method was used to establish RT-PCR amplification of mRNA transcripts of IL-2, -10, and interferon-gamma (IFN) in RNA isolated from canine peripheral blood leukocytes. Integrity of RNA isolates was ensured by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or beta-actin, IL-8, -12, and TNF were amplified from RNA isolated by various methods. Use of guanidinium-thiocyanate with and without RNA matrix gave the most consistent results. Boiling as a mean of RNA isolation was quick and easy, but the RT-PCR results were extremely variable and multiple smaller bands were observed in the agarose gel in some preparations. IL-2, -10 and IFN transcripts were amplified from RNA isolated with guanidinium-thiocyanate from leukocytes stimulated with concanavalin A. DNase-treatment of RNA isolates was necessary to assure the destruction of genomic DNA and to avoid amplification of genomic sequences. This was especially a problem when using primers for GAPDH, beta-actin, IL-12, and TNF. Lack of DNase-treatment may lead to false positive results. This may be especially a problem when amplification of so-called house-keeping genes is used as internal control for RNA integrity. These findings demonstrated that isolation of total RNA with guanidinium-thiocyanate followed by DNase-treatment gave reliable and consistent results for detection of cytokine transcripts by RT-PCR in a canine macrophage cell line and canine peripheral blood leukocytes.
Institut für Veterinär-Pathologie, Justus-Liebig-Universität Giessen, Germany.
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Fonfara
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Baumgärtner
D000199:Actins / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D016207:Cytokines / Q000302:isolation & purification*; D017931:DNA Primers / Q000737:chemistry; D004285:Dogs / Q000235:genetics* / Q000276:immunology; D004587:Electrophoresis, Agar Gel; D005188:False Negative Reactions; D005189:False Positive Reactions; D005434:Flow Cytometry / Q000662:veterinary; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D007371:Interferon-gamma / Q000302:isolation & purification; D016753:Interleukin-10 / Q000302:isolation & purification; D018664:Interleukin-12 / Q000302:isolation & purification; D007376:Interleukin-2 / Q000302:isolation & purification; D016209:Interleukin-8 / Q000302:isolation & purification; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D014409:Tumor Necrosis Factor-alpha / Q000302:isolation & purification
D000199:Actins; D000911:Antibodies, Monoclonal; D016207:Cytokines; D017931:DNA Primers; D007376:Interleukin-2; D016209:Interleukin-8; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D018664:Interleukin-12; D012313:RNA; D007371:Interferon-gamma; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases