Abstract
The inherited diseases hyperkalemic periodic paralysis and paramyotonia congenita are caused by mutations in the adult skeletal muscle sodium channel gene. To determine if differences in the expression patterns of the adult and cardiac/fetal sodium channel genes could explain some clinical features of these disorders, we developed a novel mRNA quantitation strategy called quantitative multiplex fluorescent polymerase chain reaction (QMF-PCR). This assay tests the relative levels of multiple mRNA species simultaneously using automated sequenators. We show validation of this method by competitive-PCR and RNase protection. Developmental studies of sodium channel mRNAs in humans and mice by QMF-PCR showed that the adult sodium channel mRNA quickly increased, while the cardiac/fetal sodium channel mRNA slowly decreased similarly in both limb and diaphragm muscle. We find that the adult sodium channel gene expression is predominant in fetal and neonatal muscle of both humans and mice: adult isoform mRNA concentration in fetal muscle was 8.4 x 10(-6) micrograms/micrograms of total RNA; cardiac/fetal isoform mRNA was 2.0 x 10(-6) micrograms/micrograms; and actin mRNA was 3.4 x 10(-3) micrograms/micrograms. Our results suggest that differential sodium channel gene expression correlates with age of onset of disease, but not with diaphragm involvement, in patients with hyperkalemic periodic paralysis.
Highlights
From the Departments of Molecular Genetics and Biochemistry,Human Genetics and Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
These different experimental systems generated different conclusions: RNA studies suggested that cardiadfetal sodium channel gene expression mRNAquickly increased, while the cardiaclfetal sodium predominates in fetal muscle, while protein studies suggested channel mRNA slowly decreased in both limb that there may be relatively high levels of adult, tetroxdoxinand diaphragm muscle
Singlset-rand cDNAwas synthesized Competitive PCR-A range of known amountsof RNAs produced in by incubation of 0.5-24 pg of total RNA at 42 "C for 1h in 12.5 pl of 50 vitro fromthe constructed plasmids was added to1pg of fetal skeletal mM Tris-HC1buffer containing75 mM KCl, 3 mM MgCl, 10 m~ muscle totalRNA, and single-strandcDNA synthesized by reverse trandithiothreitol, and 0.5 pg of oligo(dT) primer with 15 units of avian scriptionwith specific antisenseprimers of both mouseadultand myeloblastosisvirus reverse transcriptase(BM), 1mM each dNTPs,and cardiadfetal sodium channel genes were used as templates for PCR
Summary
Singlset-rand cDNAwas synthesized Competitive PCR-A range of known amountsof RNAs produced in by incubation of 0.5-24 pg of total RNA at 42 "C for 1h in 12.5 pl of 50 vitro fromthe constructed plasmids was added to1pg of fetal skeletal mM Tris-HC1buffer (pH 8.3) containing mM KCl, 3 mM MgCl,, 10 m~ muscle totalRNA, and single-strandcDNA synthesized by reverse trandithiothreitol, and 0.5 pg of oligo(dT) primer with 15 units of avian scriptionwith specific antisenseprimers of both mouseadultand myeloblastosisvirus reverse transcriptase(BM), 1mM each dNTPs,and cardiadfetal sodium channel genes were used as templates for PCR. Sequencingof mouse cardiadfetal and adult sodium channel geneTs he PCR products for adult and cardiadfetal sodium channel gene were was doneby cross-species PCR, as described previously [12]P.CR prim- combined and analyzed on a 6% sequencing gel by an automated seers were synthesizedfrom the cDNA sequence for the humanand/or rat quenator.
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