Universal reference method for real-time PCR gene expression analysis of preimplantation embryos

Bower, Neil Ivan, Moser, Ralf Joachim, Hill, Jonathan Robert and Lehnert, Sigrid Arabella (2007) Universal reference method for real-time PCR gene expression analysis of preimplantation embryos. BioTechniques, 42 2: 199-206.

Author Bower, Neil Ivan
Moser, Ralf Joachim
Hill, Jonathan Robert
Lehnert, Sigrid Arabella
Title Universal reference method for real-time PCR gene expression analysis of preimplantation embryos
Journal name BioTechniques   Check publisher's open access policy
ISSN 0736-6205
1940-9818
Publication date 2007
Sub-type Article (original research)
Volume 42
Issue 2
Start page 199
End page 206
Total pages 8
Place of publication New York, NY, United States
Publisher Informa Healthcare
Language eng
Formatted abstract
Real-time PCR expression profiling in individual preimplantation embryos poses two main challenges. First, the amount of RNA from blastocysts (between 100 and 200 cells) is too small to quantify, and secondly, a reference gene with stable expression across preimplantation embryos produced by different reproductive technologies is required. We have developed a method using RNA and DNA spikes that allows for accurate normalization of gene expression without the use of an internal housekeeping gene in preimplantation blastocysts. Prior to the simultaneous extraction of RNA and DNA, plant-specific RNA and DNA spikes are added to the tissue. After synthesis of cDNA, target gene transcript and the exogenous RNA spike are measured using real-time PCR. To account for differences in the number of cells in each sample, the genomic gene copies of 18S-DNA are measured by quantitative PCR and normalized to the DNA spike. While the DNA spike accounts for extraction efficiency, the 18S genomic target indicates the number of cells prior to extraction. The values obtained from normalizing the target gene to the RNA spike can be adjusted for cell number, allowing the RNA spike to be used as reference gene. This universal reference approach allows the use of an exogenous spike as a pseudo-housekeeping gene for normalization of gene expression data.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ
Additional Notes Authors prepress title: "Development of a universal reference method for quantitative real-time PCR gene expression analysis of preimplantation embryos".

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Veterinary Science Publications
 
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Created: Wed, 15 Jan 2014, 13:49:53 EST by Professor Jonathan Hill on behalf of School of Veterinary Science