Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos.

BACKGROUND: The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. RESULTS: 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 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 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 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.

Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.

BACKGROUND: Current methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost. RESULTS: This paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3), as well as their RNA transcripts in single mouse embryos and in isolated blastomeres. CONCLUSION: This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.

One-step RT-LATE-PCR for mRNA and viral RNA detection and quantification.

LATE-PCR is an optimized form of asymmetric PCR that efficiently generates high levels of single-stranded DNA amplicons. Single-stranded amplicons are advantageous because, as shown in this chapter, they can be probed at low temperature(s) with one or more probes. Based on its properties, LATE-PCR is also useful for constructing multiplex assays. Viral RNA or RNA present in cells can be detected and quantified using LATE-PCR preceded by a reverse transcription (RT) step that converts RNA into cDNA. According to the conventional two-step approach, RT is primed using nonspecific random oligonucleotides prior to performing PCR amplification in a separate step. Recently, however, the so-called "one-step" RT-PCR strategy has gained increasing popularity because all reagents needed for both reactions are added together in a single mix, thus reducing the possibility of contamination, a consideration particularly relevant to viral samples analysis. We describe below two protocols using RT-LATE-PCR and provide specific guidelines for the general application of this technique. The first protocol is devised to quantify mRNA from mouse embryos by real-time PCR; the second protocol employs end-point analysis to detect a number of different viral-like sequences in a multiplex reaction.

Heat shock memory in preimplantation mouse embryos.

To investigate the consequences of possible physiologic stress to embryos caused by in vitro fertilization procedures, we used heat shock response in preimplantation mouse embryos as a model. A heat shock "memory" was discovered that renders cleavage-stage embryos more responsive at the transcriptional level to secondary perturbation with very low doses of heat, even several cell cycles after the initial stress has occurred.

Early onset of heat-shock response in mouse embryos revealed by quantification of stress-inducible hsp70i RNA.

Heat shock response is fully established in mouse embryos at the blastocyst stage, but it is unclear when this response first arises during development. To shed light on this question, we used a single-tube method to quantify mRNA levels of the heat shock protein genes hsp70.1 and hsp70.3 (hsp70i) in individual cleavage-stage embryos that had or had not been heat-shocked. While untreated, healthy embryos contained very low copy numbers of hsp70i RNA, heat shock rapidly induced the synthesis of hundreds of hsp70i transcripts per blastomere at both the 4-cell and the 8-cell stages. In addition, we performed hsp70i measurements in embryos that had not been heat-shocked but had been very slow in developing. Quantification of hsp70i RNA and genomic DNA copy numbers in these slow-growing embryos demonstrated the presence of two distinct populations. Some of the embryos contained considerable levels of hsp70i RNA, a finding consistent with the hypothesis of endogenous metabolic stress accompanied by cell cycle arrest and delayed development. Other slow-growing embryos contained no hsp70i RNA and fewer than expected hsp70i gene copies, suggesting the possibility of ongoing apoptosis. In conclusion, this study demonstrates that mouse embryos can activate hsp70i expression in response to sub-lethal levels of stress as early as at the 4-cell stage. Our results also indicate that quantification of hsp70i DNA and RNA copy numbers may provide a diagnostic tool for embryonic health.

Laser zona drilling does not induce hsp70i transcription in blastomeres of eight-cell mouse embryos.

To assess whether zona drilling with a 1,480-nm laser induces heat shock in eight-cell embryos, we measured hsp70i RNA levels in sets of single blastomeres isolated after laser treatment of mouse embryos that had or had not been heated at 43 degrees C. Unlike heating, laser zona drilling did not stimulate hsp70i expression, even in the blastomeres closest to the laser beam, corroborating the safety of this procedure for assisted reproduction.

Developmentally-regulated changes of Xist RNA levels in single preimplantation mouse embryos, as revealed by quantitative real-time PCR.

Xist RNA localizes to the inactive X chromosome in cells of late cleavage stage female mouse embryos (Sheardown et al., 1997: Cell 91:99-107). Fluorescence in situ hybridization (FISH), however, does not quantify the number of Xist transcripts per nucleus. We have used real-time reverse transcription-polymerase chain reaction (RT-PCR) to measure Xist RNA levels in single preimplantation embryos and to establish developmental profiles in both female and male samples. The gender of each embryo was readily established based on Xist RNA levels, by counting Xist gene copies per cell, and by independent detection of the presence/absence of Sry, a Y chromosome-specific gene. Xist expression in males was found to be very low at all stages, as suggested by FISH. In contrast, female embryos contained measurable levels of Xist mRNA starting at the late 2-cell stage and rapidly accumulated Xist transcripts until morula stage. Xist RNA accumulation per embryo then reached a plateau, while cell division continued. We propose that during early cleavage high enough levels of Xist mRNA are transcribed to generate a pool of unbound molecules. This pool would serve to temporarily maintain X chromosome inactivation without additional transcription while the trophectoderm and inner cell mass (ICM) differentiate. The ICM would then loose the paternally imprinted pattern of X inactivation originally present in all embryonic cells.

Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.

Xist gene expression begins at the late 2-cell stage in female mouse embryos and by the third division results in the accumulation of an average 100 copies of Xist RNA per cell, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). In the blastocyst, the trophectoderm maintains the paternally imprinted pattern of Xist expression present during early development, while either the maternal or the paternal X chromosome can express Xist among cells of the inner mass. Fluorescent in situ hybridization (FISH) has previously established that Xist transcripts are localized on the silenced X chromosome, forming aggregates of variable dimensions in blastomeres of 8-cell embryos. This observation and the fact that Xist RNA accumulation per cell sharply decreases after morula stage raise the possibility that cells of cleaving embryos contain different levels of Xist RNA, perhaps linked to their subsequent developmental fates. We show here that Xist RNA is efficiently recovered from single blastomeres isolated from 8-cell embryos following laser zona drilling. Sexing of the samples and simultaneous quantification of Xist RNA in individual cells is achieved with a multiplex Xist/Sry real-time RT-PCR assay sensitive to the single-copy level. This analysis reveals that Xist RNA is indeed accumulated to substantially different levels in individual blastomeres of the same 8-cell embryo and that two blastomeres contain most of the molecules per embryo. These results support the conclusion that cells of the early mammalian embryo are not all functionally equivalent. Differential Xist gene expression could arise from differences in DNA methylation, or the order in which cells divide.