Quantitative RT-PCR amplification of RNA in single mouse oocytes and preimplantation embryos.
We describe a simple whole-cell method for quantitative reverse transcription (RT) PCR amplification of RNA that consistently allows the analysis of trace amounts of RNA, such as those carried by a fraction of a single mouse oocyte or preimplantation embryo, without organic extraction. The method is based on a preliminary genomic DNA digestion by DNase I in the presence of Mn++ and a subsequent RT step with rTth Reverse Transcriptase at 70 degrees C with the same buffer components, which also has the effect to irreversibly denature DNase I activity. Because of the completeness of genomic DNA digestion and RNA recovery, this procedure makes it possible to quantitatively amplify any target RNA, including those coded by intronless genes or genes whose intron-exon boundaries are unknown. By taking mRNAs of beta-actin, heat-shock protein HSP70.1 and ribosomal protein S16 as experimental models, we demonstrate the effectiveness of genomic DNA digestion by DNase I-Mn++ and of DNase I heat-denaturation and the quantitative properties of our method. We also show that this procedure is useful for transcriptional analyses during development that are hindered by paucity of biological material.
Fiorenza
M T
MT
La Sapienza University of Rome, Italy.
Mangia
F
F
D000199:Actins / Q000032:analysis / Q000235:genetics; D000818:Animals; D001755:Blastocyst / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004795:Enzyme Stability; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000235:genetics; D008345:Manganese / Q000378:metabolism; D051379:Mice; D009865:Oocytes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012269:Ribosomal Proteins / Q000032:analysis / Q000235:genetics
D000199:Actins; D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C023509:ribosomal protein S16; D008345:Manganese; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I