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

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.