An mRNA amplification procedure with directional cDNA cloning and strand-specific cRNA synthesis for comprehensive gene expression analysis.
We developed an integrated system suitable for comprehensive gene expression studies including construction and analysis of cDNA microarrays starting from a small amount of mRNA. We amplified total mRNA first as cDNA mixtures by polymerase chain reaction and then as strand-specific cRNA mixtures by in vitro transcription. These amplified cDNA and cRNA enabled determination of mRNA levels by hybridization analyses such as Southern, Northern, reverse-Northern macroarray, and cDNA microarray analyses, as well as construction of the cDNA library with a unidirectional cDNA insert. By using strand-specific cRNA derived from rat primary-cultured hepatocytes, we detected putative antisense transcripts for the metallothionein gene. cDNA microarray analysis for genes regulated by glucocorticoids and glucagon in the hepatocytes revealed that a number of genes involved in signal transduction and transcriptional regulation were up- or down-regulated. The present system is widely applicable to gene expression analysis with limited amounts of RNA samples.
Department of Biochemistry and Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan.
Satoko
S
Ohtsuka
Katsuro
K
Iwase
Masaki
M
Kato
Naohiko
N
Seki
Atsuko
A
Shimizu-Yabe
Osamu
O
Miyauchi
Eiko
E
Sakao
Masaki
M
Kanazawa
Shigenori
S
Yamamoto
Yoichi
Y
Kohno
Masaki
M
Takiguchi
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D015870:Gene Expression; D015723:Gene Library; D005934:Glucagon / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D016372:RNA, Antisense / Q000235:genetics; D018075:RNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic / Q000235:genetics
D018076:DNA, Complementary; D005938:Glucocorticoids; D016372:RNA, Antisense; D018075:RNA, Complementary; D012333:RNA, Messenger; D005934:Glucagon; D008668:Metallothionein