Abstracts 2007
March 26-30, 2007, 3rd International qPCR Symposium and Application Workshop, Freising-Weihenstephan, Germany.
Duplex RT-LATE-PCR Reveals Transcript Gradients in Sets of Single Cells Recovered from 8-Cell Mouse Embryos
Cristina Hartshorn, Odelya Hartung, and Lawrence J. Wangh
The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass (ICM) are pluripotent and express transcripts such as Oct4 RNA, which are down-regulated in the surrounding, differentiated trophectoderm (TE). Conversely, other genes are active in the TE and silenced in the ICM. These include Xist (expressed only in females) and Cdx2 (in both sexes). Prior to blastocyst formation, all these RNAs are ubiquitously found in blastomeres of embryos at the 8-cell stage. It is plausible, however, that transcript levels differ among blastomeres of the same embryo, and that these quantitative differences may presage the fate of their daughter cells. Testing this hypothesis presents numerous technical challenges because it requires simultaneous quantification of different RNAs in sets of single cells isolated from the same embryo. We have overcome these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR.
We initially constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy using both biological samples and analysis of the LATE-PCR fluorescent signals. The linear slope of these signals is a sensitive tool to establish that amplification has been achieved with comparable efficiency for all templates analyzed. The Oct4/Xist duplex was an ideal test system, because comparison of data from males and females allowed us to determine that, due to the properties of LATE-PCR, Oct4 amplification was unaffected by sex-related differences in Xist expression (females: Oct4 +, Xist ++; males: Oct4 +, Xist -).
Our results show 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. This is significant because all cells in the 8-cell embryo are believed to be developmentally equivalent. Our data also indicate that Xist and Oct4 expression levels are not correlated at this stage, although transcription of both genes is up-regulated at this time in development. We have now developed an additional assay for simultaneous measurements of Oct4 and Cdx2 RNA, in light of recent findings that these two genes are reciprocally regulated.
This work describes the first example of RT-LATE-PCR and its utility for single-tube, multiplex quantitative analysis of transcripts in single cells. Levels of different RNAs can be accurately measured independently of their relative abundance; this is not possible with symmetric PCR. The techniques illustrated here are widely applicable, for instance to gene expression analysis in stem cells and cancer cells and to preimplantation genetic diagnostics. We are also employing these strategies for multiplex quantitative end-point detection of RNA viruses.
March 19-20, 2007, Cambridge Healthtech Institute's Quantitative PCR: Replicating and Validating Success, San Diego, California.
Multiplexing, Multiprobing, Multisequencing using LATE-PCR
Lawrence J. Wangh
As reported previously LATE-PCR is an efficient form of asymmetric PCR which uses pairs of primers of unequal concentration designed such that the functional melting temperature of the Limiting Primer is equal to or greater than the functional melting temperature of the Excess Primer (TmL-TmX≥0). We are now extending this logic to construction of multiplex reactions for simultaneous amplification of sets of cDNA or genomic DNA target sequences. These multiplexed reactions generate copious levels of several single-stranded targets that can be detected in real-time or at end-point using combinations of sequence-specific probes, or mis-match tolerant probes that hybridize to variant sequences in a temperature-dependent manner. End-point ratios of such probe-target signals can then be computed as “Fluorescent Signatures”, one for each sequence variant. In addition, multiple single-stranded amplicons in a multi-plex reactions can also be sequenced one-by-one, each with a different primer, using either Dilute-'N'-Go Pyrosequencing or Dilute-‘N’-Go Dideoxy-sequencing. For instance, heteroplasmic mixtures containing mixtures of SNP’s can be identified and quantified. All of these methods are sensitive down to the level of single starting molecules. In sum, these novel methods greatly enhance the information generated from a single closed-tube reactions and promise to revolutionize PCR based in vitro diagnostics. We are currently employing these technologies for construction of diagnostic assays in the fields of infectious diseases, cancer, forensics, and gene expression.
February 27-March 2, 2007, Cambridge Healthtech Institute's Molecular Medicine Tri-Conference, San Francisco, California.
LATE-PCR and Related Technologies for Reliable Asymmetric Amplification and Characterization of Single-stranded DNA
Lawrence J. Wangh
1. The logic of LATE-PCR
2. PCR-elixirs for suppression of mispriming at all stages of amplification
3. Separation of primer annealing and probe detection
4. Quantitative end-point detection over a broad low-temperature range (60 to 25 °C)
5. Construction of fluorescent fingerprints
6. Reliable single-molecule detection using LATE-PCR and elixirs
7. Dilute-‘N’-Go sequencing following LATE-PCR
8. One-step RT-LATE-PCR
February 23-25, 2007, International Meeting on Emerging Diseases and Surveillance, Vienna, Austria.
The Rapid Detection of Influenza A and B Sub-Types, Including Avian: Using a Single-Tube LATE-PCR Assay in a Portable, Point-of-Care System
Arthur H. Reis, Jr., Cristina Hartshorn, John E. Rice, Kenneth E. Pierce, and Lawrence J. Wangh
Background: Brandeis University in collaboration with Smiths Detection is developing a highly informative in vitro diagnostic point-of-care system that will detect and discriminate between influenza subtypes, including highly pathogenic H5N1.
Methods: Our approach utilizes novel technologies for sample preparation and amplification, as well as a new portable device. Reverse transcription of RNA templates and amplification of the cDNA are both achieved using Linear-After-The-Exponential, LATE-PCR, in a closed-tube. LATE-PCR is an advanced form of asymmetric PCR which generates single-stranded products of each amplicon. The final multiplex assay is designed to simultaneously generate up to three single-stranded amplicons for each of the possible viral subtypes and a positive control. The assay is quantitative over seven orders of magnitude and is sensitive down to fewer than 10 target molecules. Novel fluorescent probes permit Quantitative End-Point Analysis, rather than real-time analysis. In addition, this assay is highly reproducible because it uses special chemistries to suppress mis-priming.
Results: The LATE-PCR influenza assay has the capacity to distinguish 15 different possible outcomes in a single closed-tube. In addition, because LATE-PCR generates single-stranded amplicons, each of the amplified products can be sent to the laboratory for immediate sequencing by a simple Dilute-‘N’-Go procedure in order to characterize new variations of specific sub-types. Specific aspects of assay results will be presented.
Conclusion: The LATE-PCR influenza assay meets the requirements of being both specific and sensitive, while rapidly detecting both current and new varieties of influenza A and B sub-types. Similar LATE-PCR assays are under development for other viral and bacterial pathogens.
Abstracts 2006
Abstracts 2005
Abstracts 2004
Abstracts 2003
