Development and validation of an accurate quantitative real time
-
Upload
moa-ren-hong -
Category
Business
-
view
217 -
download
2
Transcript of Development and validation of an accurate quantitative real time
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
2
Down
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
4
6
Sperm Ovum
PGD Program
Preimplantation Genetic Diagnosis
Evil
7
Current Methods1. Comparative genomic
hybridization (CGH)
8
Current Methods1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies
9
Current Methods1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies3. Fluorescence in situ
hybridization (FISH)
10
Current Methods1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies3. Fluorescence in situ
hybridization (FISH)
Waste Time
11
Current Methods1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies3. Fluorescence in situ
hybridization (FISH)
Chip is not cheap
12
Solution 咻咻
咻咻
13
A Brief Introduction to Quantitative PCR
14
16
MATERIALS AND METHODS
Experimental Design
17
Two-phase design
Phase IUsing the cell-lines to evaluate the system and establish baseline database.
18
Two-phase design
Phase IUsing the cell-lines to evaluate the system and establish baseline database.
Phase II
Use 71 of blastocysts to evaluate the system.
19
Catalog ID Cell Type Karyotypes Numbers GM00323 Fibroblast 46,XY 10+7
GM04610 Fibroblast 47,XX,t8 5
GM09286 Fibroblast 47,XY,t9 4
GM02948 Fibroblast 47,XY,t13 5
GM04435 Fibroblast 48,XY,t16,t21 2
AG16778 B-Lymphocyte 46,XX 3
AG16782 B-Lymphocyte 46,XY 3
GM01454 B-Lymphocyte 47,XY,t12 5
AG16777 B-Lymphocyte 47,XX,t21 5
Phase I : Cell Lines
20
Phase II : Embryos
21
qPCRStatistics
Alkaline lysis 18 cycles of multiplex amplification
Real-time PCR
22
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
23
StatisticsΔCt 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
Specific Chromosome
4 16
24
StatisticsΔCt 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
ΔCt 4 16 4 21 336
25
Establish baseline database
GM00323
26
RESULTTo evaluate the utility
27
FIGURE 1Examples of qPCR-based 24-chromosome copy number results from 5-cell samples derived from nine cell lines with previously well characterized karyotypes.
28
FIGURE 1
29
FIGURE 2
30
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
31
FIGURE 2
Chromosome-specific consistency of 99.94% (1,703/1,704) and an overall 24-chromosome diagnosis consistencyof 98.6% (70/71)
32
FIGURE 3-1Examples of (gray) single-nucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies.
33
FIGURE 3-2Examples of (gray) single-nucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies.
34
DISCUSSIONqPCR-based methodology provides the first opportunity for same-day trophectoderm biopsy 24-chromosome aneuploidy screening and fresh blastocyst transfer.
35
Back to the start …
1. Enhance embryo selection
2. Increase implantation rates
3. Reduce the incidence of miscarriage
4. Reduce the time of implantation
5. Cost-Down
PGD
qPCR
36
NO question !No comment !