Systematic Analysis of single cells by PCR Wolfgang Mann 27th March 2007, Weihenstephan
Overview AmpliGrid Technology Examples DNA Forensics Single Cell Sequencing Polar Body Diagnostics Single Cell RT PCR Advalytix AG 2007 Page 2
Challenges in qpcr Technical sample prep robust sample analysis when dealing with small volumes optimization of fluorescent dyes, development of alternative systems biochemistry of enzymes involved statistical procedures (normalization, optimization, standardization) workflow aspects, post amplification analysis, intrumentation Functional robust assay development R&D applications (sirna, etc) drug discovery Advalytix AG 2007 Page 3
towards genetic single cell analysis Analysis of small numbers of cells pools vs individuals Advalytix AG 2007 Page 4
towards genetic single cell analysis Analysis of small numbers of cells pools vs individuals Advalytix AG 2007 Page 5
towards genetic single cell analysis Examples for single or little cell numbers mouse morulae: RT-PCR analysis of single blastomeres (May et al. 2007) human polar bodies: analysis of chromosomal disorders (Hagen-Mann et al. 2005) Advalytix AG 2007 Page 6
towards genetic single cell analysis Examples for single or little cell numbers single blood cells: validation of forensic methods (Proff et al. 2006) single lymphocytes: mtdna sequence analysis (Lutz-Bonengel et al. 2006, 2007) Advalytix AG 2007 Page 7
AmpliGrid a new platform for sc amplification AmpliGrid Advalytix AG 2007 Page 8
AmpliGrid optical control of the target cell single cells on the AmpliGrid (Hoechst staining): the goal is to amplify exactly two copies of a sequence nc nc Advalytix AG 2007 Page 9
AmpliGrid robotics Main important steps: pipetting of aqueous phase (1µl) overlay with covering solution (5µl) Advalytix AG 2007 Page 10
AmpliSpeed Cycler AmpliSpeed SlideCycler non-peltier based robot compatible modular setup Advalytix AG 2007 Page 11
AmpliGrid technology Summary AmpliGrid Technology: Volume: 1µl PCR solution low cost, low thermal mass Glass slide instead of MTP improved heat conductivity Automated pipetting high throughput applications Flat surface, glass slide optical control of the target AmpliSpeed HT cycling Advalytix AG 2007 Page 12
Example: redundant PCR Improved information content by redundant PCR applying a forensic STR kit to the AmpliGrid ½vol tube PCR, conventional CE analysis DNA sample (low amount) ½vol 1µl PCR 1µl PCR 1µl PCR 1µl PCR 1µl PCR CE analysis AmpliGrid Advalytix AG 2007 Page 13
Example: redundant PCR on forensic stains 25pg of DNA: comparison of conventional vs redundant LV PCR Tube 25µL 44 LV-PCRs Typing success could be improved in 40% of the DNA samples tested Proff et al., Int. Congress Series 1288: 645-647, 2006 Redundant PCR in 1µl could be a general approach to other applications as well Advalytix AG 2007 Page 14
Example: forensic PCR Improved sensitivity on the AmpliGrid platform Comparison of different amplification geometries, subsequent sequencing mtdna amplification using primers L29/H381 Lutz-Bonengel et al., Int. J. Legal Med 121: 68-73, 2007 Advalytix AG 2007 Page 15
Example: forensic PCR Improved sensitivity on the AmpliGrid platform in order to study heteroplasmy of mitochondrial DNA FACS on Ampli Grid PCR + sequencing in 1µl Lutz-Bonengel et al., in prep Advalytix AG 2007 Page 16
Example: single cell sequencing Simple workflow for single cell sequencing lymphocyte preparation by FACS (48 cells in < 30s) PCR amplification of mtdna adding ExoSAPit in order to get rid of PCR primers adding cycle sequencing kit (including seqeuncing primer) cycle sequencing transfer into MTP apply to CE and read out In a systematic study Lutz-Bonengel et al (2007) could show: 96 % of heteroplasmy occur within tissues 4 % occur within cells or within mitochondria Diagnostics: clonal expansion of mtdna haplotypes in leukemia patients can be analysed by mtdna analysis (Yao et al. 2007) Advalytix AG 2007 Page 17
Example: PCR on single sequences DNA Typing of single cells with forensic STR kits (ABI) different numbers of single cells as a model for distinct copy numbers systematic study on FACS sorted cells (lymphocytes) Proff et al. 2006; drawbacks: FACS sort not optimized no nucleus staining Basic data for single cell analysis from forensic stains Advalytix AG 2007 Page 18
Example: human polar body analysis The goal is to detect presence or absence of all human chromosomes in polar bodies after sample preparation by glass capillary. Advalytix AG 2007 Page 19
Example: human polar body analysis Workflow: Sample preparation under the microscope (optical control) Deposition of polar bodies onto the AmpliGrid Transfer to the molecular lab (optical control of the sample) PCR analysis Polar body analysis is an example for an application with: A limited number of cells is to be analysed Each of the cells has to be analysed There is no second shot AmpliGrid has turned out to be an excellent storage device Optical control is the advantage for quality control of the workflow Advalytix AG 2007 Page 20
Polar body analysis: the test The test routine is as follows: Deposition of the single cell onto the AmpliGrid Overall amplification of the genome in 1µl (reducing complexity) Distribution of this amplicon to a number of multiplex PCRs Detection of products in HT mode (PAA, LiCor, etc) 5 markers / chromosome designed test result for single cell amplification Advalytix AG 2007 Page 21
Polar body analysis: results PCR results on polar body analysis Advalytix AG 2007 Page 22
Expanding the number of markers / genome Polar body analysis: results Advalytix AG 2007 Page 23
principle of ABC Amplification Based Counting PCR success rate = # of successful sub-pcrs total # of sub-pcrs ABC hinges on the concept of redundant sub-pcrs which amplify sub-sequences located within a sequence whose copy number is to be counted The average success rate of a set of sub-pcrs and the copy number of a starting sequence (template) correspond very reliably: for a given PCR protocol, drop-outs occur once the copy number falls below a fixed threshold ( dropout regime ) ABC comprises a range of diagnostic tools to exploit this fundamental insight Advalytix AG 2007 Page 24
applications in human genetics Cause disease Copy number deviation causes medical condition Examples Turner syndrome (45,X0) Trisomies: 13, 18, 21 Deletions: Cri-du-Chat (5p), Wolf-Hirschhorn (4p) HER2 (breast cancer) P53 (tumor suppressor) Mark disease Copy number change in a noncoding section of DNA Presents pre-disposition to disease or indicates a medical condition Examples STRs as markers for LOH (loss of heterocygozity) -5p -17q Microsatellites (MIN): HNPCC (hereditary nonpolyposis colon cancer) Single cell analysis is needed in IVF, fetal cells in maternal blood, studying mosacism, CTC, etc... Advalytix AG 2007 Page 25
Example: RT-PCR RNA amplification on the AmpliGrid has been shown to be very sensitive. As an example, a 308bp fragment was amplified from a 3000b transcript using RT-PCR. As little as 10 copies can be detected. LS: MW standard 10 5 : 100.000 RNA copies 10 4 : 10.000 RNA copies 10 3 : 1000 RNA copies 10 2 : 100 RNA copies 10: 10 RNA copies 1: 1 RNA copy 0: negative cotrol Advalytix AG 2007 Page 26
Example: RT-PCR on CD4+ cells Cells have been FACS sorted after staining with an anti CD4-antibody Expression of CD4 mrna was shown in some of the single cells 310bp RT PCR product specific for CD4 mrna combination of immunophenotyping and genetic analysis of the identical cell: a very new field to exploit Advalytix AG 2007 Page 27
Example: complex RT PCR on mouse embryos Analysis of gene expression of single mouse embryos a set of genes has been chosen that are all expressed in the mouse embryo: DNA modification genes encoding DNA methyltransferases Dnmt3a, Dnmt3b, Dnmt1, Dnmt3l methyl-cpg binding domain proteins Mbd1, Mbd2, Mbd3 and Mbd4 DNA repair proteins Polb, Apex1, Lig1, Lig3 (active demethylation by base excision) control genes H3.3, Pou5f1 Advalytix AG 2007 Page 28
Example: complex RT PCR on mouse embryos Experimental set up: sensitivity test on total RNA analysis of morulae analysis of single blastocystes Multiplex RT PCR on 1000pg of total RNA, 14 genes, 12 replicates (left / right lane: molecular size marker) Advalytix AG 2007 Page 29
Example: complex RT PCR on mouse embryos Analysis of total mouse morulae A1, A2,... : different mouse morulae PBS, (EC), H2O: negative controls 50bp: molecular weight marker Advalytix AG 2007 Page 30
Example: complex RT PCR on mouse embryos Analysis of single mouse blastocystes M A1 A4 A6 A8 A9 A10 B1 B4 PCR RT PBS PBS (EC) H 2 0 A1, A2,..B1,. : different mouse blastocysts PBS, (EC), H2O, PCR, RT: negative controls M: molecular weight marker Preliminary work will be followed by systematic analysis and semiquantitative analysis of products (May et. al, 2007, in prep) Advalytix AG 2007 Page 31
summary AmpliGrid is a new platform for amplification reactions (PCR/RT-PCR) It has been designed for single cell use Optical features of the chip allow to qualify the target cells Different characteristics might be combined (immunophenotyping + PCR) AmpliGrid allows high throughput, systematic analysis of single cells Even other ideas are on the way: culture the cell on chip, stress the cell,.. Advalytix AG 2007 Page 32
contributions Advalytix thanks the following partners: GSF, Neuherberg Gerichtsmedizinisches Institut, Universität Köln Gerichtsmedizinischs Institut, Universität Freiburg Humangenetisches Institut, Universität Mainz Advalytix AG 2007 Page 33