Development and optimization of a fluorescent differential display PCR system for studying bovine embryo development in vitro.
Differential display is a widely used methodology to identify genes that are differentially expressed in biological samples. We developed a new protocol for the amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg mRNA) from preimplantation bovine embryos. The cDNAs generated with an anchor primer, associated with a universal sequence, were amplified with an arbitrary primer and a single fluorescently labeled primer. Amplification products were easily visualized with a fluorescence scanner, without the need for radioisotopes. Nineteen isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the recognition and identification of gene transcripts involved in bovine embryonic physiology.
Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brasil.
Paula
P
Ripamonte
Giovana Krempel
GK
Merighe
Yeda Fumie
YF
Watanabe
Alexandre Rodrigues
AR
Caetano
Flávio Vieira
FV
Meirelles
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics; D047108:Embryonic Development / Q000235:genetics*; D020224:Expressed Sequence Tags; D005307:Fertilization in Vitro / Q000379:methods*; D005453:Fluorescence; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012333:RNA, Messenger