Quantification of llama inflammatory cytokine mRNAs by real-time RT-PCR.
We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF) alpha were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1alpha, IL-1beta, IL-6 and TNFalpha were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.
Odbileg
Raadan
R
Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido 060-0818, Japan.
Konnai
Satoru
S
Usui
Tatsufumi
T
Ohashi
Kazuhiko
K
Onuma
Misao
M
D000818:Animals; D052160:Benzothiazoles; D002161:Camelids, New World / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D017931:DNA Primers; D003959:Diamines; D004305:Dose-Response Relationship, Drug; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D052160:Benzothiazoles; D016207:Cytokines; D017931:DNA Primers; D003959:Diamines; D008070:Lipopolysaccharides; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I