Application of cDNA arrays to monitor mRNA profiles in single preimplantation mouse embryos.
Array technology is a widely used tool for gene expression profiling in various biological systems. However, the application of this method to mammalian preimplantation embryos is limited by the small amount of mRNA that can be extracted from a single embryo, which is not sufficient for array analysis. Here we report a protocolfor the rapid global amplification of embryonic mRNA that permits the generation of expression profiles from single murine blastocysts. The approach combines global PCR and 77 RNA polymerase amplification and allows the preparation of labeled, amplified RNA for array hybridization from single murine blastocysts containing approximately 1.5 pg mRNA in less than 12 h. We demonstrate that this amplification procedure is highly reproducible and does not bias original relative mRNA levels. Signal patterns from various embryonic stages of murine development revealed marked differences in mRNA expression that were in accordance with previously published data. We found genes known to be involved in embryonic apoptosis expressed at different levels in individual murine day 3.5 blastocysts. This technique can thus be used to assess embryonic viability and investigate molecular mechanisms of embryonic development.
Institute for Animal Science (FAL), Neustadt, Germany.
T
T
Brambrink
P
P
Wabnitz
R
R
Halter
R
R
Klocke
J
J
Carnwath
W
W
Kues
C
C
Wrenzycki
D
D
Paul
H
H
Niemann
D000818:Animals; D001755:Blastocyst; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice / Q000196:embryology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D012333:RNA, Messenger