Quantitation of metallothionein mRNA by RT-PCR and chemiluminescence.
A general procedure has been developed for the determination of mRNA expression by reverse transcription polymerase chain reaction (RT-PCR), over a wide concentration range, with quick quantitation of amplified products by luminescence. The discriminating power of this approach is the specific hybridization of PCR product to ruthenium-labeled oligonucleotide probe(s). This method is sensitive enough to detect increases in the formation of PCR product by the 6th cycle. The accumulation of PCR product was successfully modeled with a recursive relationship. This procedure was capable of accurately determining starting template copies over a 9-log dynamic range, with a sensitivity limit of 10(2) copies. Inclusion of an mRNA internal standard (identical to amplified template except for a 6-bp deletion) corrected variabilities in the reverse transcriptase as well as PCR, allowing for the expression of data as mRNA copy number/micrograms total tissue RNA. This procedure was used to detect changes in levels of winter flounder (Pleuronectes americanus) liver metallothionein mRNA. Liver metallothionein mRNA levels ranged from 1.0 x 10(6) copies/micrograms total tissue RNA in control samples to 1.0 x 10(9) copies/micrograms total tissue RNA in samples treated with Cd (a known metallothionein mRNA inducer).
Jessen-Eller
K
K
University of Connecticut, Groton.
Picozza
E
E
Crivello
J F
JF
D000818:Animals; D001483:Base Sequence; D015342:DNA Probes; D005432:Flounder / Q000235:genetics; D008099:Liver / Q000737:chemistry; D008163:Luminescent Measurements; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase
D015342:DNA Probes; D012333:RNA, Messenger; D008668:Metallothionein; D012194:RNA-Directed DNA Polymerase