Reprogramming, Screening and Validation of iPSCs and
Terminally Differentiated Cells using the
qBiomarker PCR Array System...
Outline of Webinar
What are induced pluripotent Stem Cells (iPS Cells or iPSCs)?
Methods for creating iPSCs
Screening and ...
Why iPSCs? What can they be used for?

iPSCs are derived from somatic cells and are not subjected to moral/policy issues
t...
What are inducible pluripotent Stem Cells (iPS or iPSCs?)

Most common technique for iPSCs Creation
Reprogram a somatic ce...
Basic Workflow in creating iPSCs

-5-

Sample & Assay Technologies
Basic Workflow in creating iPSCs

-6-

Sample & Assay Technologies
Basic Workflow in creating iPSCs

Cell types:
Fibroblasts
Keratinocytes
pancreatic beta cells
Hepatocytes
Any somatic cell...
Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???

Cell types:...
Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???

Reprogrammi...
Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???

Reprogrammi...
Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???

Delivery Me...
Pluripotency Validation

How Can we validate the pluripotency of our iPSCs?
Chimeria Formation
Teratoma Formation
Biochemi...
Pluripotency Validation

How Can we validate the pluripotency of our iPSCs?
Chimeria Formation
Teratoma Formation
Biochemi...
Using qPCR As a Screening and Validation Technology
qPCR: (Real-Time PCR)
Able to accurately identify and provide quantita...
Key Considerations in qPCR Assay Design

Cycling Conditions

Perfect
qPCR
Assay
Design Algorithm

Mastermix

Functional Va...
Key Considerations in qPCR Assay Design

Cycling Conditions

Perfect
qPCR
Assay
Design Algorithm

Mastermix

Functional Va...
qBiomarker PCR Arrays

qBiomarker iPSC Colony Screening

qBiomarker Screening PCR Arrays

Samples

1
2
3
4
5
6
7
8

DNMT3B...
qBiomarker PCR Arrays

qBiomarker iPSC Colony Screening

qBiomarker Screening PCR Arrays

Samples

1
2
3
4
5
6
7
8

DNMT3B...
qBiomarker PCR Arrays

qBiomarker iPSC Colony Screening

qBiomarker Screening PCR Arrays

Samples

1
2
3
4
5
6
7
8

DNMT3B...
qBiomarker PCR Arrays

qBiomarker iPSC Colony Screening

qBiomarker Screening PCR Arrays

Samples

1
2
3
4
5
6
7
8

DNMT3B...
qBiomarker PCR Arrays

qBiomarker iPSC Colony Screening

qBiomarker Screening PCR Arrays

Samples

1
2
3
4
5
6
7
8

DNMT3B...
qBiomarker PCR Arrays

qBiomarker iPSC Colony Screening

qBiomarker Screening PCR Arrays

Samples

1
2
3
4
5
6
7
8

DNMT3B...
qBiomarker PCR Arrays

qBiomarker iPSC Colony Screening

qBiomarker Screening PCR Arrays

Samples

1
2
3
4
5
6
7
8

DNMT3B...
qBiomarker Screening and Validation PCR Arrays
2 different formats depending on number of Biomarkers
qBiomarker Screening ...
qBiomarker PCR Arrays Biological Content
Biomarkers are selected using multi-step process
1. Text mining and literature re...
How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample

Isolate RNA using QIAGEN RNeasy
.

cDNA Synthesis (...
How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample

Isolate RNA using QIAGEN RNeasy
.

cDNA Synthesis (...
How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample

Isolate RNA using QIAGEN RNeasy
.

cDNA Synthesis (...
How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample

Isolate RNA using QIAGEN RNeasy
.

cDNA Synthesis (...
How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample

Isolate RNA using QIAGEN RNeasy
.

cDNA Synthesis (...
Melting Curve Analysis
Single Dissociation Peaks for Every Gene Assay

• Example of QC criteria for
every PCR Assay
manufa...
Melting Curve Analysis
Single Dissociation Peaks for Every Gene Assay

• Example of QC criteria for
every PCR Assay
manufa...
Melting Curve Analysis
Single Dissociation Peaks for Every Gene Assay

• Example of QC criteria for
every PCR Assay
manufa...
Compatible Instrumentation: 96- & 384-Well Formats

96-Well Blocks: 7000, 7300, 7500, 7700
FAST 96-Well Blocks: 7500, 7900...
Data Analysis is Simple, Quick and Predictive
An integral part of ALL qBiomarker PCR Arrays
Each Pathway has a specific Da...
Screening iPS Cells for Pluripotency
Experiment:
Created 6 different iPS cell lines from fibroblasts following 3 weeks of
...
Screening iPS Cells for Pluripotency
Experiment:
Created 6 different iPS cell lines from fibroblasts following 3 weeks of
...
Screening iPS Cells for Pluripotency
Experiment:
Created 6 different iPS cell lines from fibroblasts following 3 weeks of
...
Example of Graphs
∆ Ct Value Norm alized by GAPDH

∆ Ct Value Norm alized by GAPDH

18

18

16

16
Fib

14

DNM T3B

12

i...
Application Data
Determining The Pluripotent Status of iPSCs:

.

One of the problems facing iPSC researchers is
spontaneo...
iPSC Pluripotency Validation

- 41 -

Sample & Assay Technologies
iPSC Pluripotency Validation

- 42 -

Sample & Assay Technologies
Summary
Reprogramming
qBiomarker Expression Lentivirus
qBiomarker Expression Plasmids

- 43 -

Sample & Assay Technologies
Summary
Reprogramming
qBiomarker Expression Lentivirus
qBiomarker Expression Plasmids
iPS Cell Induction
•iPSC colony scre...
Summary
Reprogramming
qBiomarker Expression Lentivirus
qBiomarker Expression Plasmids
iPS Cell Induction
•iPSC colony scre...
Summary
qBiomarker PCR Arrays
Laboratory validated performance and biological content
Available for any qPCR instrument
Co...
Thank you for attending our Webinar!

Questions, Comments or Suggestions?
Please type all questions into the “Questions” B...
Upcoming SlideShare
Loading in …5
×

I psc and stem cell 2013

369 views

Published on

0 Comments
2 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
369
On SlideShare
0
From Embeds
0
Number of Embeds
4
Actions
Shares
0
Downloads
21
Comments
0
Likes
2
Embeds 0
No embeds

No notes for slide

I psc and stem cell 2013

  1. 1. Reprogramming, Screening and Validation of iPSCs and Terminally Differentiated Cells using the qBiomarker PCR Array System Sample & Assay Technologies
  2. 2. Outline of Webinar What are induced pluripotent Stem Cells (iPS Cells or iPSCs)? Methods for creating iPSCs Screening and Validation iPSCs using qPCR The qBiomarker PCR Array System Application Data Screening of iPSC for Pluripotency Summary, Questions and Answers -2- Sample & Assay Technologies
  3. 3. Why iPSCs? What can they be used for? iPSCs are derived from somatic cells and are not subjected to moral/policy issues that surround embryonic stem cells. Not necessary to destroy or create embryos Not subjected to Federal/State/Political funding issues Uses of iPSCs in Biomedical Research: Basic Research into pluripotency and differentiation Applied Research into Disease Specific model systems Translational Research Uses in regenerative medicine -3- Sample & Assay Technologies
  4. 4. What are inducible pluripotent Stem Cells (iPS or iPSCs?) Most common technique for iPSCs Creation Reprogram a somatic cell by the over expression of key regulatory factors: Yamanaka Factors: Oct4, Sox2, KLF4, c-MYC Thomson Factors: Oct4, Sox2, Nanog, Lin28 -4- Sample & Assay Technologies
  5. 5. Basic Workflow in creating iPSCs -5- Sample & Assay Technologies
  6. 6. Basic Workflow in creating iPSCs -6- Sample & Assay Technologies
  7. 7. Basic Workflow in creating iPSCs Cell types: Fibroblasts Keratinocytes pancreatic beta cells Hepatocytes Any somatic cell? -7- Sample & Assay Technologies
  8. 8. Basic Workflow in creating iPSCs Factors: Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog siRNA, small molecules, ??? Cell types: Fibrobalsts Pancreatic beta cells Hepatocytes Any somatic cell? -8- Sample & Assay Technologies
  9. 9. Basic Workflow in creating iPSCs Factors: Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog siRNA, small molecules, ??? Reprogramming (2-3 weeks) Cell types: Fibrobalsts Pancreatic beta cells Hepatocytes Any somatic cell? -9- Sample & Assay Technologies
  10. 10. Basic Workflow in creating iPSCs Factors: Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog siRNA, small molecules, ??? Reprogramming (2-3 weeks) Cell types: Fibrobalsts Pancreatic beta cells Hepatocytes Any somatic cell? - 10 - Sample & Assay Technologies
  11. 11. Basic Workflow in creating iPSCs Factors: Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog siRNA, small molecules, ??? Delivery Methods: Infection (lentivirus) Transfection Electroporation Reprogramming (2-3 weeks) Cell types: Fibrobalsts Pancreatic beta cells Hepatocytes Any somatic cell? How do we validate Pluripotency? - 11 - Sample & Assay Technologies
  12. 12. Pluripotency Validation How Can we validate the pluripotency of our iPSCs? Chimeria Formation Teratoma Formation Biochemical Assays ? ? ? Differentiation and Morphology (Microscopy) Biomarkers using qPCR - 12 - Sample & Assay Technologies
  13. 13. Pluripotency Validation How Can we validate the pluripotency of our iPSCs? Chimeria Formation Teratoma Formation Biochemical Assays ? ? ? Differentiation and Morphology (Microscopy) Biomarkers using qPCR - 13 - Sample & Assay Technologies
  14. 14. Using qPCR As a Screening and Validation Technology qPCR: (Real-Time PCR) Able to accurately identify and provide quantitation for nucleic acids Small amounts of sample needed Can look at multiple genes simultaneously SABiosciences’ Solution to qPCR is the PCR Array Collection of Wet-Bench Validated SYBR Green qPCR Assays Biological Content for understanding biological pathway or defining mechanism of action Standardized for any qPCR instrument Data Analysis and Biological Interpretation qBiomarker PCR Arrays use laboratory validated samples to define both a predictive Biomarker set of genes and data analysis algorithm to clearly differentiate between a Biological process or define disease. - 14 - Sample & Assay Technologies
  15. 15. Key Considerations in qPCR Assay Design Cycling Conditions Perfect qPCR Assay Design Algorithm Mastermix Functional Validation (Wet-Bench Testing) Ensures all parameters are optimized - 15 - Sample & Assay Technologies
  16. 16. Key Considerations in qPCR Assay Design Cycling Conditions Perfect qPCR Assay Design Algorithm Mastermix Functional Validation (Wet-Bench Testing) Ensures all parameters are optimized - 16 - Perfect qPCR Assay Re-validate Assay with New MM Sample & Assay Technologies
  17. 17. qBiomarker PCR Arrays qBiomarker iPSC Colony Screening qBiomarker Screening PCR Arrays Samples 1 2 3 4 5 6 7 8 DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC 8 samples per Plate - 17 - 8 predictive Biomarkers 1 Normalization Gene GDC (Genomic DNA Control) RTC (Reverse Transcription Control) PPC (Positive PCR Control) Sample & Assay Technologies
  18. 18. qBiomarker PCR Arrays qBiomarker iPSC Colony Screening qBiomarker Screening PCR Arrays Samples 1 2 3 4 5 6 7 8 DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC 8 samples per Plate - 18 - 8 predictive Biomarkers 1 Normalization Gene GDC (Genomic DNA Control) RTC (Reverse Transcription Control) PPC (Positive PCR Control) Sample & Assay Technologies
  19. 19. qBiomarker PCR Arrays qBiomarker iPSC Colony Screening qBiomarker Screening PCR Arrays Samples 1 2 3 4 5 6 7 8 DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC 8 samples per Plate - 19 - 8 predictive Biomarkers 1 Normalization Gene GDC (Genomic DNA Control) RTC (Reverse Transcription Control) PPC (Positive PCR Control) Sample & Assay Technologies
  20. 20. qBiomarker PCR Arrays qBiomarker iPSC Colony Screening qBiomarker Screening PCR Arrays Samples 1 2 3 4 5 6 7 8 DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC 8 samples per Plate - 20 - 8 predictive Biomarkers 1 Normalization Gene GDC (Genomic DNA Control) RTC (Reverse Transcription Control) PPC (Positive PCR Control) Sample & Assay Technologies
  21. 21. qBiomarker PCR Arrays qBiomarker iPSC Colony Screening qBiomarker Screening PCR Arrays Samples 1 2 3 4 5 6 7 8 DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC 8 samples per Plate - 21 - 8 predictive Biomarkers 1 Normalization Gene GDC (Genomic DNA Control) RTC (Reverse Transcription Control) PPC (Positive PCR Control) Sample & Assay Technologies
  22. 22. qBiomarker PCR Arrays qBiomarker iPSC Colony Screening qBiomarker Screening PCR Arrays Samples 1 2 3 4 5 6 7 8 DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC 8 samples per Plate - 22 - 8 predictive Biomarkers 1 Normalization Gene GDC (Genomic DNA Control) RTC (Reverse Transcription Control) PPC (Positive PCR Control) Sample & Assay Technologies
  23. 23. qBiomarker PCR Arrays qBiomarker iPSC Colony Screening qBiomarker Screening PCR Arrays Samples 1 2 3 4 5 6 7 8 DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC 8 samples per Plate - 23 - 8 predictive Biomarkers 1 Normalization Gene GDC (Genomic DNA Control) RTC (Reverse Transcription Control) PPC (Positive PCR Control) Sample & Assay Technologies
  24. 24. qBiomarker Screening and Validation PCR Arrays 2 different formats depending on number of Biomarkers qBiomarker Screening PCR Arrays 8 samples/plate iPSC Colony Screening Yamanaka Factors Thomson Factors qBiomarker Validation PCR Arrays 4 samples/plate iPSC Pluripotency Validation - 24 - Sample & Assay Technologies
  25. 25. qBiomarker PCR Arrays Biological Content Biomarkers are selected using multi-step process 1. Text mining and literature research 2. Large gene list developed 3. Biological Samples assayed through collaboration 4. Predicative Gene Signature based on Results 5. Companion Data Analysis Algorithm trained and tested - 25 - Sample & Assay Technologies
  26. 26. How do qBiomarker PCR Arrays Work? Isolate Total RNA from Your Sample Isolate RNA using QIAGEN RNeasy . cDNA Synthesis (C-03 kit) – – . Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates Multiple Samples per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data (FREE) 10 minutes from Raw Ct values to interpretation of Biology and Fold Change results - 26 - Sample & Assay Technologies
  27. 27. How do qBiomarker PCR Arrays Work? Isolate Total RNA from Your Sample Isolate RNA using QIAGEN RNeasy . cDNA Synthesis (C-03 kit) – – . Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates Multiple Samples per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data (FREE) 10 minutes from Raw Ct values to interpretation of Biology and Fold Change results - 27 - Sample & Assay Technologies
  28. 28. How do qBiomarker PCR Arrays Work? Isolate Total RNA from Your Sample Isolate RNA using QIAGEN RNeasy . cDNA Synthesis (C-03 kit) – – . Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates Multiple Samples per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data (FREE) 10 minutes from Raw Ct values to interpretation of Biology and Fold Change results - 28 - Sample & Assay Technologies
  29. 29. How do qBiomarker PCR Arrays Work? Isolate Total RNA from Your Sample Isolate RNA using QIAGEN RNeasy . cDNA Synthesis (C-03 kit) – – . Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates Multiple Samples per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data (FREE) 10 minutes from Raw Ct values to interpretation of Biology and Fold Change results - 29 - Sample & Assay Technologies
  30. 30. How do qBiomarker PCR Arrays Work? Isolate Total RNA from Your Sample Isolate RNA using QIAGEN RNeasy . cDNA Synthesis (C-03 kit) – – . Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates Multiple Samples per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data (FREE) 10 minutes from Raw Ct values to interpretation of Biology and Fold Change results - 30 - Sample & Assay Technologies
  31. 31. Melting Curve Analysis Single Dissociation Peaks for Every Gene Assay • Example of QC criteria for every PCR Assay manufactured by SABiosciences • Single peak dissociation curves • Single gel bands of predicted size - 31 - Sample & Assay Technologies
  32. 32. Melting Curve Analysis Single Dissociation Peaks for Every Gene Assay • Example of QC criteria for every PCR Assay manufactured by SABiosciences • Single peak dissociation curves • Single gel bands of predicted size - 32 - Sample & Assay Technologies
  33. 33. Melting Curve Analysis Single Dissociation Peaks for Every Gene Assay • Example of QC criteria for every PCR Assay manufactured by SABiosciences • Single peak dissociation curves • Single gel bands of predicted size - 33 - Sample & Assay Technologies
  34. 34. Compatible Instrumentation: 96- & 384-Well Formats 96-Well Blocks: 7000, 7300, 7500, 7700 FAST 96-Well Blocks: 7500, 7900HT FAST 384-Well Block: 7900HT StepOnePlus iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384 Opticon, Opticon 2, Chromo 4 Mastercycler ep realplex 2/2S/4/4S LightCycler 480 TP-800 Mx3000p, Mx3005p, Mx4000p qBiomarker PCR Arrays are guaranteed when using qBiomarker qPCR SYBR Green MasterMix - 34 - Sample & Assay Technologies
  35. 35. Data Analysis is Simple, Quick and Predictive An integral part of ALL qBiomarker PCR Arrays Each Pathway has a specific Data Analysis Template Interprets Gene Expression Changes into Biological Process . Are my iPSCs Pluripotent? What is the expression level of endogenous reprogramming Factors? Exogenous? Have my iPSCs undergone spontaneous differentiation? Are my cardiomyocytes or neurons terminally differentiated? - 35 - Sample & Assay Technologies
  36. 36. Screening iPS Cells for Pluripotency Experiment: Created 6 different iPS cell lines from fibroblasts following 3 weeks of reprogramming. Which cell lines are totally pluripotent and worth using? iPS Colony Screening Delta Ct Values are interpreted - 36 - Sample & Assay Technologies
  37. 37. Screening iPS Cells for Pluripotency Experiment: Created 6 different iPS cell lines from fibroblasts following 3 weeks of reprogramming. Which cell lines are totally pluripotent and worth using? iPS Colony Screening Delta Ct Values are interpreted - 37 - Sample & Assay Technologies
  38. 38. Screening iPS Cells for Pluripotency Experiment: Created 6 different iPS cell lines from fibroblasts following 3 weeks of reprogramming. Which cell lines are totally pluripotent and worth using? iPS Colony Screening Delta Ct Values are interpreted Drop iPSC 1 and iPSC 2 from further experiments. - 38 - Sample & Assay Technologies
  39. 39. Example of Graphs ∆ Ct Value Norm alized by GAPDH ∆ Ct Value Norm alized by GAPDH 18 18 16 16 Fib 14 DNM T3B 12 iPSC 1 12 DPPA4 iPSC 2 10 iPSC 3 iPSC 4 8 ∆ Ct ∆ Ct 14 GDF3 10 LEFTY1 NANOG 8 iPSC 5 6 iPSC 6 4 ESC PODXL POU5F1 4 2 6 ZFP42 2 0 0 DNM T3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 ZFP42 Fib iPSC 1 iPSC 2 iPSC 3 Genes iPSC 5 iPSC 6 ESC Sam ples Fold Change Comparing to Control Sample Fold Change Comparing to Control Sample 5.0 5.0 4.5 4.5 4.0 iPSC 1 3.5 iPSC 2 3.0 iPSC 3 2.5 iPSC 4 2.0 iPSC 5 1.5 iPSC 6 1.0 ESC F o ld ch an g e (L o g 10) F old ch ang e (L o g10) iPSC 4 0.5 DNMT3B 4.0 DPPA4 3.5 GDF3 3.0 LEFTY1 2.5 NANOG 2.0 PODXL 1.5 POU5F1 1.0 ZFP42 0.5 0.0 DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 0.0 ZFP42 iPSC 1 Genes iPSC 2 iPSC 3 iPSC 4 iPSC 5 iPSC 6 ESC Samples - 39 - Sample & Assay Technologies
  40. 40. Application Data Determining The Pluripotent Status of iPSCs: . One of the problems facing iPSC researchers is spontaneous differentiation and loss of pluripotency during culture and passage conditions. Are my IPSCs still pluripotent? ESC A.1 iPSC #1 iPSC #2 IMR-90 iPSC Valdation PCR Array - 40 - Sample & Assay Technologies
  41. 41. iPSC Pluripotency Validation - 41 - Sample & Assay Technologies
  42. 42. iPSC Pluripotency Validation - 42 - Sample & Assay Technologies
  43. 43. Summary Reprogramming qBiomarker Expression Lentivirus qBiomarker Expression Plasmids - 43 - Sample & Assay Technologies
  44. 44. Summary Reprogramming qBiomarker Expression Lentivirus qBiomarker Expression Plasmids iPS Cell Induction •iPSC colony screening •iPSC Pluripotency Validation •Yamanaka Factors •Thomson Factors •Reprogramming Factors Expression - 44 - Sample & Assay Technologies
  45. 45. Summary Reprogramming qBiomarker Expression Lentivirus qBiomarker Expression Plasmids iPS Cell Induction •iPSC colony screening •iPSC Pluripotency Validation •Yamanaka Factors •Thomson Factors •Reprogramming Factors Expression iPS Cell Differentiation •Embryoid Body •Neuronal Differentiation •Cardiomyocytes Differentiation - 45 - Sample & Assay Technologies
  46. 46. Summary qBiomarker PCR Arrays Laboratory validated performance and biological content Available for any qPCR instrument Companion Data Analysis for interpretation of results Applications: Screening or Validation of iPS Cell inductions Validation of Cardiomyocyte and Neuronal Differentiation Complete System for Reprogramming, Screening and Validating iPS Cells - 46 - Sample & Assay Technologies
  47. 47. Thank you for attending our Webinar! Questions, Comments or Suggestions? Please type all questions into the “Questions” Box. - 47 - Sample & Assay Technologies

×