Se ha denunciado esta presentación.
Utilizamos tu perfil de LinkedIn y tus datos de actividad para personalizar los anuncios y mostrarte publicidad más relevante. Puedes cambiar tus preferencias de publicidad en cualquier momento.
Development and validation of an accurate
quantitative real-time polymerase chain reaction–
based assay for human blastocy...
Down

2
The Reason We Start
1. Enhance embryo selection
2. Increase implantation rates

3. Reduce the incidence of miscarriage
4. ...
4
Preimplantation Genetic Diagnosis
Sperm Ovum

PGD Program
Evil

6
Current Methods
1. Comparative genomic
hybridization (CGH)

7
Current Methods
1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies

8
Current Methods
1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies
3. Fluorescence in situ
hybridi...
Current Methods
1. Comparative genomic
hybridization (CGH)

Waste Time

2. SiNP microarray technologies
3. Fluorescence in...
Current Methods
1. Comparative genomic
hybridization (CGH)

Chip is not cheap
2. SiNP microarray technologies
3. Fluoresce...
Solution

咻
咻
咻
咻

12
A Brief Introduction to Quantitative PCR

13
14
MATERIALS
AND METHODS
Experimental Design

16
Two-phase design
Phase I
Using the cell-lines to evaluate
the system and establish
baseline database.

17
Two-phase design
Phase I
Using the cell-lines to evaluate
the system and establish
baseline database.
Phase II
Use 71 of b...
Phase I : Cell Lines
Catalog ID
GM00323
GM04610
GM09286
GM02948
GM04435
AG16778
AG16782
GM01454
AG16777

Cell Type

Karyot...
Phase II : Embryos

20
qPCR
Statistics

Alkaline lysis

18 cycles of
multiplex amplification

Real-time PCR
21
Statistics
ΔCt was calculated from the average ∆Ct of the
16 reactions targeting a specific Chromosome
minus the average ∆...
Statistics
ΔCt was calculated from
the average ∆Ct of the 16
reactions targeting a
specific Chromosome
minus the average ∆...
Statistics
ΔCt was calculated from
the average ∆Ct of the 16
ΔCt a 4
reactions targeting
specific Chromosome
minus the ave...
Establish baseline database

GM00323

25
RESULT
To evaluate the utility

26
FIGURE 1
Examples of qPCR-based
24-chromosome copy
number results from 5cell samples derived from
nine cell lines with
pre...
FIGURE 1

28
FIGURE 2

29
FIGURE 2

97.6% reliability of obtaining a
diagnosis and a100% level of
consistency of chromosome-specific
(n =984) and 24...
FIGURE 2

Chromosome-specific consistency
of 99.94% (1,703/1,704) and an
overall 24-chromosome diagnosis
consistencyof 98....
FIGURE 3-1
Examples of (gray) singlenucleotide polymorphism
microarray–
and (white) qPCR-based
24-chromosome copy
number r...
FIGURE 3-2
Examples of (gray) singlenucleotide polymorphism
microarray–
and (white) qPCR-based
24-chromosome copy
number r...
DISCUSSION
qPCR-based methodology
provides the first opportunity
for same-day trophectoderm
biopsy 24-chromosome
aneuploid...
Back to the start …
1. Enhance embryo selection
2. Increase implantation rates

PGD

3. Reduce the incidence of miscarriag...
NO question !
No comment !

36
Próxima SlideShare
Cargando en…5
×

1

Compartir

Descargar para leer sin conexión

Development and validation of an accurate quantitative real time

Descargar para leer sin conexión

Libros relacionados

Gratis con una prueba de 30 días de Scribd

Ver todo

Audiolibros relacionados

Gratis con una prueba de 30 días de Scribd

Ver todo

Development and validation of an accurate quantitative real time

  1. 1. Development and validation of an accurate quantitative real-time polymerase chain reaction– based assay for human blastocyst comprehensive chromosomal aneuploidy screening Author : Nathan R. Treff (Ph.D), et al. Fertility and Sterility (IF: 4.174) VOL. 97 NO. 4 / APRIL 2012 Speaker : Hung, Mau-Ren 1
  2. 2. Down 2
  3. 3. The Reason We Start 1. Enhance embryo selection 2. Increase implantation rates 3. Reduce the incidence of miscarriage 4. Reduce the time of implantation 3
  4. 4. 4
  5. 5. Preimplantation Genetic Diagnosis Sperm Ovum PGD Program Evil 6
  6. 6. Current Methods 1. Comparative genomic hybridization (CGH) 7
  7. 7. Current Methods 1. Comparative genomic hybridization (CGH) 2. SiNP microarray technologies 8
  8. 8. Current Methods 1. Comparative genomic hybridization (CGH) 2. SiNP microarray technologies 3. Fluorescence in situ hybridization (FISH) 9
  9. 9. Current Methods 1. Comparative genomic hybridization (CGH) Waste Time 2. SiNP microarray technologies 3. Fluorescence in situ hybridization (FISH) 10
  10. 10. Current Methods 1. Comparative genomic hybridization (CGH) Chip is not cheap 2. SiNP microarray technologies 3. Fluorescence in situ hybridization (FISH) 11
  11. 11. Solution 咻 咻 咻 咻 12
  12. 12. A Brief Introduction to Quantitative PCR 13
  13. 13. 14
  14. 14. MATERIALS AND METHODS Experimental Design 16
  15. 15. Two-phase design Phase I Using the cell-lines to evaluate the system and establish baseline database. 17
  16. 16. Two-phase design Phase I Using the cell-lines to evaluate the system and establish baseline database. Phase II Use 71 of blastocysts to evaluate the system. 18
  17. 17. Phase I : Cell Lines Catalog ID GM00323 GM04610 GM09286 GM02948 GM04435 AG16778 AG16782 GM01454 AG16777 Cell Type Karyotypes Numbers Fibroblast 46,XY 10+7 Fibroblast Fibroblast 47,XX,t8 47,XY,t9 5 4 Fibroblast Fibroblast 47,XY,t13 48,XY,t16,t21 5 2 B-Lymphocyte 46,XX 3 B-Lymphocyte B-Lymphocyte 46,XY 47,XY,t12 3 5 B-Lymphocyte 47,XX,t21 5 19
  18. 18. Phase II : Embryos 20
  19. 19. qPCR Statistics Alkaline lysis 18 cycles of multiplex amplification Real-time PCR 21
  20. 20. Statistics ΔCt was calculated from the average ∆Ct of the 16 reactions targeting a specific Chromosome minus the average ∆ Ct of all of the 336 reactions targeting all of the remaining autosomes 22
  21. 21. Statistics ΔCt was calculated from the average ∆Ct of the 16 reactions targeting a specific Chromosome minus the average ∆ Ct of all of the 336 reactions targeting all of the remaining autosomes 4 16 Specific Chromosome 23
  22. 22. Statistics ΔCt was calculated from the average ∆Ct of the 16 ΔCt a 4 reactions targeting specific Chromosome minus the average ∆ Ct of all of the 336 reactions targeting all of the remaining autosomes 16 4 21 336 24
  23. 23. Establish baseline database GM00323 25
  24. 24. RESULT To evaluate the utility 26
  25. 25. FIGURE 1 Examples of qPCR-based 24-chromosome copy number results from 5cell samples derived from nine cell lines with previously well characterized karyotypes. 27
  26. 26. FIGURE 1 28
  27. 27. FIGURE 2 29
  28. 28. FIGURE 2 97.6% reliability of obtaining a diagnosis and a100% level of consistency of chromosome-specific (n =984) and 24-chromosome copy number (n =41) assignments 30
  29. 29. FIGURE 2 Chromosome-specific consistency of 99.94% (1,703/1,704) and an overall 24-chromosome diagnosis consistencyof 98.6% (70/71) 31
  30. 30. FIGURE 3-1 Examples of (gray) singlenucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies. 32
  31. 31. FIGURE 3-2 Examples of (gray) singlenucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies. 33
  32. 32. DISCUSSION qPCR-based methodology provides the first opportunity for same-day trophectoderm biopsy 24-chromosome aneuploidy screening and fresh blastocyst transfer. 34
  33. 33. Back to the start … 1. Enhance embryo selection 2. Increase implantation rates PGD 3. Reduce the incidence of miscarriage 4. Reduce the time of implantation 5. Cost-Down qPCR 35
  34. 34. NO question ! No comment ! 36
  • ok7260678

    Mar. 12, 2015

Vistas

Total de vistas

766

En Slideshare

0

De embebidos

0

Número de embebidos

7

Acciones

Descargas

7

Compartidos

0

Comentarios

0

Me gusta

1

×