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1.  Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos 
BMC Biotechnology  2007;7:87.
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.
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.
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.
PMCID: PMC2246118  PMID: 18067662
2.  Two-temperature LATE-PCR endpoint genotyping 
BMC Biotechnology  2006;6:44.
In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay.
Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles.
SNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions.
PMCID: PMC1698914  PMID: 17144924
3.  Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell 
BMC Biotechnology  2005;5:2.
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.
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.
This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.
PMCID: PMC546192  PMID: 15649321

Results 1-3 (3)