sybr-green-i and Peripheral-Nervous-System-Diseases

Mutations and altered gene dosage of the peripheral myelin protein (PMP22) gene in chromosome 17p11.2-12 are the main causes for hereditary neuropathies, accounting for approximately 70% of all cases. Patients with duplication of the PMP22 develop Charcot-Marie-Tooth disease type 1A (CMT1A) and deletion of one PMP22 allele leads to hereditary neuropathy with liability to pressure palsy (HNPP). Twenty patients with CMT1A, 17 patients with HNPP, and 18 normal family members and 28 normal controls were studied by real-time quantitative PCR using SYBR Green I on the ABI 7700 Sequence Detection System. The copy number of the PMP22 gene was determined by the comparative threshold cycle method and the albumin was used as a reference gene. The PMP22 duplication ratio ranged from 1.45 to 2.06 and the PMP22 deletion ratio ranged from 0.42 to 0.64. The PMP22 ratio in normal controls, including normal family members, ranged from 0.85 to 1.26. No overlap was found between patients with CMT1A or patients with HNPP and normal controls. This method is fast, highly sensitive, specific, and reproducible in detecting PMP22 duplication and deletion in CMT1A and HNPP patients, respectively.

Topics: Benzothiazoles; Charcot-Marie-Tooth Disease; Chromosomes, Human, Pair 17; Diamines; Family Health; Female; Fluorescent Dyes; Gene Deletion; Gene Duplication; Hereditary Sensory and Motor Neuropathy; Humans; Male; Membrane Proteins; Organic Chemicals; Paralysis; Peripheral Nervous System Diseases; Quinolines; Reverse Transcriptase Polymerase Chain Reaction

The P2X receptor is a receptor-gated cationic channel that responds to ATP. The quantification of P2X mRNA expression in dorsal root ganglion (DRG) provides important information for neuropathic pain studies. We developed a rapid and sensitive external-standard-based real-time quantitative PCR assay for the quantification of mRNA of P2X receptors in mouse tissue samples. The assay uses a double-stranded DNA fluorescent dye, SYBR Green I, to continuously monitor product formation with a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). To establish the quantitative PCR amplification in a wide range of target transcripts, optimum parameters of primer sequences, concentrations of primers and/or templates, and PCR thermal protocols were experimentally determined. We also tested the reliability of this method in established experimental murine models, which were made by ligation or cutting down of the sciatic nerve. The parameters defined in this assay should be applicable to the quantification of other types of pain models and other tissue samples of mouse.

Topics: Animals; Benzothiazoles; Diamines; Disease Models, Animal; Fluorescent Dyes; Ganglia, Spinal; Mice; Nerve Crush; Neuralgia; Neurons, Afferent; Organic Chemicals; Peripheral Nervous System Diseases; Polymerase Chain Reaction; Quinolines; Receptors, Purinergic P2; Receptors, Purinergic P2X; RNA, Messenger; Sciatic Nerve