Background The formation of two distinctive cell lineages in preimplantation mouse

Background The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically 950912-80-8 supplier equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. Conclusion This paper describes the first example of multiplex RT-LATE-PCR and its utility, when 950912-80-8 supplier combined with PurAmp sample preparation, for quantitative analysis of transcript levels in single cells. With this technique, copy numbers of different RNAs can be accurately measured independently from their relative abundance in a cell, a goal Rabbit polyclonal to RABAC1 that cannot be achieved using symmetric PCR. The technique illustrated in this work is relevant to a wide array of applications, such as stem cell and cancer cell analysis and preimplantation genetic diagnostics. Background Accurate quantification of multiple target sequences by real-time polymerase chain reaction (PCR) has been proven difficult to achieve, particularly for measuring numbers of RNA transcripts, rather than of DNA copies [1]. In fact, while different gene sequences are represented in the genome in similar numbers (one or two copies, depending on the chromosomal location and the possible presence of mutations), transcript levels of different genes can vary widely. Moreover, changes in gene expression are often rapid and transient in response to stimuli, stress, or cellular events such as cell division and cell differentiation. In order to detect meaningful variations in transcript numbers it is, thus, necessary to measure them in series of single cells rather than in cell cohorts where individual differences could be lost to “background noise [2].” From all of the above considerations it follows that a convenient and reliable experimental approach, sensitive enough to measure RNA copy numbers 950912-80-8 supplier in individual cells, is essential for multiplex quantification of gene expression in biological systems. We have recently developed an entirely single-tube method to measure mRNA levels in individual cells (“PurAmp”) [3]. For 950912-80-8 supplier that and a previous study [4] we co-amplified and simultaneously quantified 950912-80-8 supplier RNA and DNA copies of the Xist and the Sry genes in mouse embryos and blastomeres. These two genes were chosen because they have sexually distinct patterns of expression in the early embryo. Female cells contain two copies of the Xist gene, one on each X-chromosome, and high levels of Xist transcripts but lack the Sry-bearing Y-chromosome, while cells from early-stage male embryos have a single unexpressed copy of the Xist gene on the X-chromosome and a single unexpressed copy of Sry on the Y-chromosome. Hence, only Xist themes (cDNA+ genomic DNA) were amplified from woman samples, while male embryos constantly generated equivalent numbers of Xist and Sry amplicons. In these studies we were, therefore, never faced with the more common and problematic scenario of having to concurrently quantify unequal amounts of different target sequences, a technical challenge of great general interest. In fact, standard symmetric PCR is not very easily utilized in this scenario due to the exponential nature of the reaction. With this method, abundant.