Quantification of interleukin-6 mRNA in wallerian degeneration by competitive reverse transcription polymerase chain reaction.
Following peripheral nerve injury, cytokines might be critically involved in regulating the cellular response within the lesioned nerve but are frequently difficult to measure due to their low abundancy. Competitive quantitative RT-PCR assays are potentially useful in quantifying cytokine mRNAs in small tissue samples but frequently do not fulfill the theoretical demands on proportional amplification of natural and standard sequences. Using this technique, we constructed standard curves for rat interleukin (IL)-6 mRNA with strictly proportional slopes and shifts when varying natural and standard RNA concentrations. A strong and transient increase in IL-6 mRNA was found in both nerve stumps 12 h after rat sciatic nerve transection, with low expression already in uninjured rat sciatic nerves. IL-6 might be involved in regulating the cellular response to peripheral nerve injury, and standard curves obtained by independent variation of standard and natural RNA are helpful in detecting and controlling experimental variables and pitfalls in competitive RT-PCR assays, thus increasing their reliability and reproducibility.
Bourde
O
O
Department of Neurology, University of Würzburg, Germany.
Kiefer
R
R
Toyka
K V
KV
Hartung
H P
HP
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D015850:Interleukin-6 / Q000235:genetics*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D012584:Sciatic Nerve / Q000378:metabolism; D014855:Wallerian Degeneration / Q000235:genetics*
D017931:DNA Primers; D015850:Interleukin-6; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger