A simple nested RT-PCR method for quantitation of the relative amounts of multiple cytokine mRNAs in small tissue samples.
It is difficult to quantitate cytokine mRNA profiles in small human tissue specimens obtained by a needle biopsy, even using standard RT-PCR methods, because the amount of mRNA in the specimens is very small. To address this problem, we developed highly sensitive, quantitative, nested RT-PCR techniques to evaluate the expression of multiple cytokine mRNAs in synovial specimens obtained by needle biopsy. To reduce effects of variation of initial RNA concentrations, cDNA from each target RNA sample was normalized, using a simplified competitive PCR method, to the levels of beta-actin cDNA. The first and the second (nested) PCR were performed in the same tube to prevent contamination. The number of PCR-product bands, evident on polyacrylamide gel electrophoresis, was used to quantitate the relative amounts of target cDNA. Using our methods, it was possible to evaluate, in a single synovial tissue specimen obtained by needle biopsy, the relative amounts of mRNAs for 10 cytokines (TNF-alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-10, IL-12 p40, IL-13, IL-15, IFN-gamma) and CD3 delta chain. Our methods are particularly valuable if there are multiple target mRNAs, numerous samples, or if the amounts of mRNAs are limited. The methods are applicable to a wide variety of tissues and target mRNAs.
Kotake
S
S
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA.
Schumacher
H R
HR
Wilder
R L
RL
D001172:Arthritis, Rheumatoid / Q000503:physiopathology; D001707:Biopsy, Needle / Q000379:methods; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers; D015870:Gene Expression; D006801:Humans; D007717:Knee; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D013582:Synovial Fluid / Q000737:chemistry
D016207:Cytokines; D017931:DNA Primers; D012333:RNA, Messenger