Detection of TNF and TNF receptor mRNA in cells and tissues.
We describe a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique for the quantification of the messenger RNA of human and murine tumor necrosis factor alpha (TNFalpha) and related receptors. This protocol can be adapted for blood, peripheral blood lymphocytes, or other tissues. We propose a dot-blot technique which, if properly set up, is fast and quantitatively reliable. We describe two different detection protocols employing either radioactive or, alternatively for laboratories that cannot or do not want to use radioactivity, fluorescent-labeled probes. We also describe our calculations for relative quantification, based on the use of a positive control sample that becomes the reference value used to compare experimental samples. These protocols have as their aim to provide a flexible tool that can be employed in several different human and murine experimental settings by laboratories with different equipment.
Furlan
Roberto
R
Department of Neurology, San Raffaele Scientific Institute, Milan, Italy.
Salani
Giuliana
G
Bergami
Alessandra
A
Martino
Gianvito
G
D000818:Animals; D001345:Autoradiography; D001671:Biochemistry / Q000379:methods*; D019793:Fluorescein / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D012313:RNA; D019793:Fluorescein