Construction of cDNA bank from biopsy specimens for multiple gene analysis of cancer.
We constructed a "cDNA bank" of human colorectal cancer and surrounding normal tissues with our unique mRNA assay system. Total nucleic acids extracted from patients' tissues were applied to 96-well plates, where poly(dT) sequences of oligonucleotides were immobilized. After hybridization, the cDNA was reverse-transcribed on the plate with the captured mRNA as a template, followed by synthesis of double-stranded (ds) cDNA. The resulting sense cDNA was removed from the plate, then used in PCR for analysis of various genes. The sense strand of the cDNA was repeatedly synthesized by using the immobilized antisense cDNA as a template even from plates used once and stored at 4 degrees C for as long as 6 months. Furthermore, the results of PCR could be easily compared among different specimens if the same amount of total mRNA were applied to the plate for the ds cDNA synthesis. This demonstrated that the cDNA bank constructed from clinical materials provides almost unlimited supplies of cDNA for multiple gene analysis of cancer.
Ishikawa
T
T
Second Department of Surgery, Yokohama City University, School of Medicine, Japan.
Ichikawa
Y
Y
Miura
Y
Y
Momiyama
M
M
Keller
C
C
Koo
K
K
Akitaya
T
T
Shimada
H
H
Mitsuhashi
M
M
D000199:Actins / Q000235:genetics; D000328:Adult; D001706:Biopsy; D015179:Colorectal Neoplasms / Q000235:genetics*; D018076:DNA, Complementary / Q000096:biosynthesis; D015723:Gene Library; D016758:Genes, jun; D006801:Humans; D009693:Nucleic Acid Hybridization; D009955:Ornithine Decarboxylase / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D013698:Templates, Genetic
D000199:Actins; D018076:DNA, Complementary; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D009955:Ornithine Decarboxylase