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Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue


Application Guide ........................................................................................................................2

I. Description ................................................................................................................................2

II. Product Components ...............................................................................................................2

III. Materials to be Supplied by the User ...................................................................................2

IV. Protocol for Extraction of DNA from FFPE tissue ...............................................................3

V. Protocol for GenomePlex Whole Genome Amplification from FFPE tissue ........................4

VI. Quantification of Amplified Products ...................................................................................5

VII. Purification of Amplified Products ......................................................................................5



Appendix ......................................................................................................................................6

Application Data ..........................................................................................................................6

References ...................................................................................................................................7

Contact Information ................. ....................................................................................................7


Application Guide

I. Description
Archived Formalin-fixed, Paraffin-embedded (FFPE) tissue samples are invaluable resources for profiling gene expression and studying a variety of diseases. Since the archived DNA is precious material and usually available in limited quantities, amplification of the samples is essential. However, amplifying the FFPE tissue can be a difficult task due to the damaged template that results from the archiving process. This protocol provides a convenient method to amplify and purify genomic DNA from FFPE tissue. The methods described are completed using the GenElute Mammalian Genomic DNA Miniprep Kit, the GenomePlex WGA kit, and GenElute PCR Clean-up Kit.

GenomePlex is a Whole Genome Amplification (WGA) method that allows the researcher to generate a representative, approximate ~500-fold amplification of genomic DNA. The amplification yield is dependent on the purity and amount of starting material. The kit utilizes a proprietary amplification technology based upon random fragmentation of genomic DNA and conversion of the resulting small fragments to PCR-amplifiable OmniPlex Library molecules flanked by universal priming sites. The OmniPlex library is then PCR amplified using universal oligonucleotide primers and a limited number of cycles. This technology maintains the genetic representation with concordance >99.8% in genotyping results from comparing genomic DNA and GenomePlex amplified DNA1. In addition to SNP genotyping, downstream applications also include performing TaqMan assays and BeadArray analysis.

II. Product Components
GenElute Mammalian Genomic DNA Miniprep Kit (G1N10)
GenomePlex WGA kit (WGA1)
GenElute PCR Clean-Up Kit (NA1020)


III. Materials to be Supplied by the User
FFPE tissue
Xylene
JumpStart Taq DNA Polymerase, Product Code D9307
Ethanol (absolute) Product Code E7023
37 C water bath or heating block
55 C water bath or heating block
70 C water bath or heating block
Microcentrifuge (with rotor for 2ml tubes)
Molecular Biology Reagent Water, Product Code W4502


IV. Protocol for Extraction of DNA from FFPE tissue
GenElute Mammalian Genomic DNA Miniprep Kit

1. Place a small section (20mg) of paraffin-embedded tissue in a 2 ml microcentrifuge tube.
2. Add 1200 l of xylene and vortex for 30 seconds.
3. Centrifuge at full speed for 5 minutes at room temperature.
4. Remove supernatant by pipettting. Do not remove any of the pellet.
5. Add 1200 l of ethanol to the pellet to remove the residual xylene, mix by vortexing.
6. Centrifuge at full speed for 5 minutes at room temperature.
7. Carefully remove the ethanol by pipetting. Do not remove any of the pallet.
8. Repeat steps 5-7 one more time.
9. Incubate the open microcentrifuge tube at 37 C for 10-15 minutes to remove any residual ethanol by evaporation.
10. Digest Tissue. Resuspend the tissue pellet in 180 l of Lysis Solution T.
11. Add 20 l of proteinase K, mix by vortexing. Incubate at 55 C (overnight) or until the tissue is completely lysed. Vortex occasionall y during incubation.
12. Optional Rnase treatment. If residual RNA is a concern add 20 l of RNase A solution and incubate at room temperature for 2 minutes.
13. Lyse cells. Vortex for 15 seconds. Add 200 l of Lysis Solution C to the sample. Vortex thoroughly as a homogenous mixture is essential for efficient lysis. Incubate at 70 C for 10 minutes.
14. Column preparation. Add 500 l of the Column Preparation Solution to each pre-assembled GenElut MiniPrep Binding Column and centrifuge at 12,000g for 1 minute.
15. Prepare for binding. Add 200 l of ethanol to the lysed sample and mix by vortexing.
16. Load lysate. Transfer the entire contents of the sample tube into the treated binding column from step 14. Centrifuge at >6,500g for 1 minute. Discard the collection tube containing the flow-through liquid and place the binding column in a new 2 ml collection tube.
17. First wash. Prior to first use, dilute the Wash Solution Concentrate with ethanol as described under preparation instructions. Add 500 l of Wash Solution to the binding column and centrifuge for 1 minute at >6,500g. Discard the collection tube containing flow-through liquid and place the binding column in a new 2 ml collection tube.
18. Second wash. Add another 500 l of Wash Solution to the binding column and centrifuge for 3 minutes at maximum speed (12,000-18,000g) to dry the binding column. It is crucial that binding column is free of ethanol before eluting DNA off the column. Centrifuge the column for an additional minute if residual ethanol is visible. Finally, discard the collection tube containing the flow through liquid and place the bindi ng column in a new 2 ml collection tube.
19. Elute DNA. Pipette 200 l of the Elution Solution directly into the center of the binding column and incubate at room temperature for 5 minutes. Centrifuge for 1 minute at >6,500g to elute the DNA.
20. Store DNA samples at -20C.

Note: This protocol can be performed without usage of xylene starting with step number 10. As a result of omitting xylene treatment step the amount of DNA will decrease by approximately 50% when compared to the protocol carried with a xylene step.


V. Protocol for GenomePlex Whole Genome Amplification from FFPE tissue
Performed with GenomePlex Whole Genome Amplification Kit (WGA1)

Fragmentation
1. Prepare DNA solution of 1 ng/l from the FFPE DNA
2. Add 1 l of 10X Fragmentation Buffer to 10 l DNA (1 ng/l) in a PCR tube.
3. Place the tube in a thermal cycler at 95 C for EXACTLY 4 minutes. Note, the incubation is time sensitive and any deviation may alter results.
4. Immediately cool the sample on ice and centrifuge briefly.

Library Preparation
5. Add 2 l of 1x Library Preparation Buffer.
6. Add 1 l of Library Stabilization Solution.
7. Mix thoroughly and place in thermal cycler at 95 C for 2 minutes.
8. Cool the sample on ice and centrifuge briefly.
9. Add 1 l Library Preparation Enzyme, mix thoroughly, and centrifuge briefly.
10. Place sample in thermal cycler and incubate as follows:
16 C for 20 minutes
24 C for 20 minutes
37 C for 20 minutes
75 C for 5 minutes
4 C hold
11. Remove samples from thermal cycler and centrifuge briefly. Samples may be amplified immediately or stored at - 20 C up to three days.

Amplification
12. Add the following reagents to the entire 15 l reaction:
7.5 l 10x Amplification Master Mix
47.5 l Nuclease Free Water
5.0 l JumpStart Taq DNA Polymerase (12.5 units)

13. Mix thoroughly, centrifuge briefly, and begin thermocycling:

Initial Denaturation 95 C for 3 minutes

Perform 14 cycles as follows:
Denature 95 C for 15 seconds
Anneal/Extend 65 C for 5 minutes

14. After cycling is complete, maintain the reactions at 4 C or store at 20 C until ready for analysis or purification.

VI. Quantification of Amplified Products
The amount of GenomePlex Whole Genome Amplification Kit products can be detected with or without purification. For the highest quality samples of DNA we strongly recommend cleaning up the samples after amplification. The amplified products can be measured with the PicoGreen dsDNA Quantitation Assay (Molecular Probes Inc. Product # P-7589). Another method of detecting the amplified products is spectrophotometric absorption (OD260) on a NanoDrop instrument.


VII. Purification of Amplified Products
Performed with GenElute PCR Clean-Up Kit (NA1020)

1. Insert a GenElute Miniprep Binding Column (with a blue o-ring) into a provided collection tube, if not already assembled. Add 0.5 ml of the Column Preparation Solution to each miniprep column and centrifuge at 12,000 x g for 30 seconds to
1 minute. Discard the eluate.

Note: The Column Preparation Solution maximizes binding of the DNA to the membrane resulting in more consistent yields.

2. A dd 5 volumes of Binding Solution to 1 volume of the PCR reaction and mix. For example, add 500 l of Binding Solution to 100 l of the PCR reaction. Transfer the solution into the binding column. Centrifuge the column at maximum speed (12,000-16,000 Xg) for 1 minute. Discard the eluate, but retain the collection tube.

3. Replace the binding column into the collection tube. Apply 0.5 ml of diluted Wash Solution to the column and centrifuge at maximum speed for 1 minute. Discard the eluate, but retain the collection tube.

Note: Be sure to add ethanol to the Wash Solution Concentrate prior to first time use. See Preparation Instructions.
4. Replace the column into the collection tube. Centrifuge the column at maximum speed for 2 minutes, without any additional wash solution, to remove excess ethanol. Discard any residual eluate as well as the collection tube.

5. Transfer the column to a fresh 2 ml collection tube. Apply 50 l of Elution Solution or water to the center of each column. Incubate at room temperature for 1 minute.

Note: When eluting with water, make sure that the pH of the water is between 5.5 and 8.5. Elution may also be performed using the Elution Solution diluted 10-fold with water.

6. To elute the DNA, centrifuge the column at maximum speed for 1 minute. The PCR amplification product is now present in the eluate and is ready for immediate use or storage at 20 C.


Appendix

Application Data

GenomePlex, a WGA kit, provides excellent representation and unsurpassed yield of the desired DNA without generating unnecessary primer assembly products.

DNA extracted from FFPE rat liver tissu e was amplified using the Genomeplex WGA kit and competitor kits starting with 0ng, 10ng or 100ng input FFPE DNA. The Sigma-Aldrich kit provided no amplification product with 0ng of input DNA (as shown above), while competitor kits resulted in primer assembly products with 0ng of input DNA.
Each of the WGA reactions were subjected to quantitative PCR (45 cycles) using 12 different primer sets. This data shows the targeted genes were more efficiently amplified (as shown by the number of cycles minus Ct values) with the GenomePlex kit as compared to the competitor kits.




References
1. Barker, D. L., et al. Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel. Genomic Research, 14, 901-7 (2004).

2. Gribble, S., et al. Chromosome paints from single copies of chromosomes. Chromosome Research, 12, 143-51 (2004).

3. Thorstenson, Y. R., et al. An Automated Hydrodynamic Process for Controlled, Unbiased DNA Shearing. Genome Research, 8, 848-855 (1998).

Contact Information
For technical assistance please contact:
Technical Service
(800) 325-5832

To learn more about GenomePlex WGA technology visit: www.sigmaaldrich.com

GenomePlex is a registered trademark of Rubicon Genomics, Inc.
GenomePlex WGA technology patent pending.
GenElute and Jumpstart are trademarks of Sigma-Aldrich, Inc.
Nanodrop is a registered trademark of Nanodrop Technologies, Inc.
PicoGreen is a trademark of Molecular Probes, Inc.
BeadArray is a Trademark of Illumina , Inc.
TaqMan is a registered trademark of Roche Molecular Systems
Taq antibody licensed for in vitro research use under U.S. Patent No. 5,338,671 and 5,587,287, and corresponding patents in other countries.
This product is sold under license from Roche Molecular Systems, Inc. and Applied Biosystems.

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