An improved quantitative RT-PCR fluorescent method for analysis of gene transcripts in the STS-65 space shuttle experiment.
We describe a reverse transcription polymerase chain reaction (RT-PCR) technique using fluorescent dUTP to examine changes in mRNA level in samples. In this procedure, the amplified product is identified by the fluorescent detection system in an automated DNA sequencer, and if the MW of the DNA/RNA or the fluorescent dye is different, several samples can be analyzed in a single lane. The basis for this technique is similar to that of radiolabeled methods, and we applied this technique for the comparison of the expression level of the rat c-myc gene in osteoblasts exposed to microgravity and unit gravity conditions. Using the fluorescent- and radiolabeled methods, the level of rat c-myc mRNA were compared quantitatively and the results demonstrated that the c-myc expression level was not altered by microgravity. Therefore, this fluorescent RT-PCR technique is useful for gene expression analysis particularly when the samples are limited, such as in space flight experiments. The method also allows for rapid assessment of mRNA changes in many samples simultaneously.
Akiyama
H
H
Biological Sciences Department, Toray Research Center, Inc., Kamakura, Japan.
Kanai
S
S
Hirano
M
M
Kumei
Y
Y
Shimokawa
H
H
Katano
H
H
Hara
E
E
Song
S
S
Mukai
C
C
Nagaoka
S
S
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D015870:Gene Expression; D016259:Genes, myc; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D010006:Osteoblasts / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013026:Space Flight; D014893:Weightlessness
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases