Novel approach to quantitative reverse transcription PCR assay of mRNA component in autopsy material using the TaqMan fluorogenic detection system: dynamics of pulmonary surfactant apoprotein A.
A novel approach to quantitative reverse transcription (RT)-PCR assay of mRNA component using fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 sequence detection system) was developed for autopsy materials. Pulmonary surfactant apoprotein A (SP-A) mRNA from a cadaveric lung was quantitated in real-time. The target SP-A gene and the endogenous reference of glyceraldehyde-3-phosphate (GAPDH) were amplified in the same tube, and an amount of the target was normalized to the reference. This assay had a high reproducibility and discrimination even in forensic autopsy materials up to 96 h postmortem. Elevated SP-A expressions were determined in some cases. This system without post-PCR sample handling would be a very useful tool in pathological diagnosis and DNA analysis.
Ishida
K
K
Department of Legal Medicine, Osaka City University Medical School Asahi-machi 1-4-3, Abeno, 545-8585, Osaka, Japan. ishida@med.osaka-cu.ac.jp
Zhu
B L
BL
Maeda
H
H
D000293:Adolescent; D000328:Adult; D000368:Aged; D001344:Autopsy; D002423:Cause of Death; D002648:Child; D016002:Discriminant Analysis; D005260:Female; D005986:Glyceraldehyde 3-Phosphate / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008875:Middle Aged; D011180:Postmortem Changes; D011510:Proteolipids / Q000235:genetics*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D013997:Time Factors
D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D012333:RNA, Messenger; D005986:Glyceraldehyde 3-Phosphate; D019914:Taq Polymerase