Stripping protocol for Affymetrix tiling gene chips

doi:10.1038/nprot.2008.240

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

Stripping protocol for Affymetrix tiling gene chips

Hoi-Hung Cheung, Wai-Yee Chan, Tin-Lap Lee

Hoi-Hung Cheung

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Wai-Yee Chan

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Tin-Lap Lee

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

DOI:

10.1038/nprot.2008.240

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Tiling microarrays integrate the genome in an unbiased fashion and provide a much higher genomic resolution compared to tradition microarrays. Affymetrix tiling microarray platform is one of the most popular tiling array platforms available, and has been widely applied in the studies of transcriptome, transcriptional and epigenetic regulations. Similar to all Affymetrix gene chip families, the tiling microarrays are designed for one time use only.

Various stripping strategies have been described in other microarray platforms. Here we describe a method for reusing the Affymetrix tiling gene chips by simple stripping procedures.

Stripping buffer: 1% SDS.

Wash buffer A: Non-Stringent Wash Buffer (6X SSPE, 0.01% Tween-20) (1 L).

Storage buffer: 0.06X SSPE.

Pre-hybridization buffer 2X: (16.6% 12X MES, 35.4% 5M NaCl, 8% 0.5M EDTA, 0.2% 10% Tween-20)

Scanner: GeneChip® Scanner 3000 7G.

Fluidics: GeneChip® Fluidics Station 450.

Hybridization Oven: GeneChip® Hybridization Oven 645.

Pre-warm the stripping buffer at 65°C.
Pre-warm the hybridization oven at 65°C.
Fill the tiling chip with 200 μl pre-warmed stripping buffer.
Place the chip in hybridization oven at 65°C. Rotate 20 min at 60 rpm.
Remove stripping buffer. Strip again with another 200 μl of stripping buffer for 10 min.
Repeat step 4 for one more time.
Remove the stripping buffer and fill with 200 μl of wash buffer A to wash. Place the chip in the hybridization oven at 65°C. Rotate 5 min at 60 rpm.
Remove the wash buffer and repeat step 6 twice.
Fill with 250 μl storage buffer. Scan chip to check stripping efficiency.
Scan the chip to check the scanned image.
If no signal is detected on the image, proceed to hybridize with the target (re-probe).
Change the oven temperature to 45°C.
Replace the storage buffer with pre-hybridization buffer. Place the chip in the hybridization oven at 45°C. Rotate 5 min at 60 rpm.
If not re-probed immediately, the chip can be stored at 4°C overnight.

1 hour

The use of fresh probes for re-probing is suggested.

If the scanned image is not clean, repeat the stripping step 3 for another 10 min.

The correlation coefficient between the first hybridization (without stripping) and re-hybridization (stripped) should be above 0.9. The range we have is around 0.96.

Kate McInnerney

commented on 06 April, 2009

In tests, this protocol also worked exceptionally well with Affy expression arrays, with little loss of signal intensity or gain of background. After three hybridizations (two strippings) the chip cassettes begin to fail, with sample leakage from the cassette face. Decreased stripping temps might be possible for cRNA hybs.

commented on 30 June, 2011

good

Kate McInnerney

commented on 06 April, 2009

commented on 30 June, 2011

good

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Subject Areas

Biotechnology

Biochemistry

Method Article

Biotechnology

Biochemistry

Stripping protocol for Affymetrix tiling gene chips

Hoi-Hung Cheung, Wai-Yee Chan, Tin-Lap Lee

Hoi-Hung Cheung

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Wai-Yee Chan

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Tin-Lap Lee

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Version History

CURRENT STATUS: Posted

Version 1

Posted 10 Dec, 2008

MetricsComments: 4HTML Views: ...

Subject Areas

Biotechnology

Biochemistry

DOI:

10.1038/nprot.2008.240

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Various stripping strategies have been described in other microarray platforms. Here we describe a method for reusing the Affymetrix tiling gene chips by simple stripping procedures.

Stripping buffer: 1% SDS.

Wash buffer A: Non-Stringent Wash Buffer (6X SSPE, 0.01% Tween-20) (1 L).

Storage buffer: 0.06X SSPE.

Pre-hybridization buffer 2X: (16.6% 12X MES, 35.4% 5M NaCl, 8% 0.5M EDTA, 0.2% 10% Tween-20)

Scanner: GeneChip® Scanner 3000 7G.

Fluidics: GeneChip® Fluidics Station 450.

Hybridization Oven: GeneChip® Hybridization Oven 645.

Pre-warm the stripping buffer at 65°C.
Pre-warm the hybridization oven at 65°C.
Fill the tiling chip with 200 μl pre-warmed stripping buffer.
Place the chip in hybridization oven at 65°C. Rotate 20 min at 60 rpm.
Remove stripping buffer. Strip again with another 200 μl of stripping buffer for 10 min.
Repeat step 4 for one more time.
Remove the stripping buffer and fill with 200 μl of wash buffer A to wash. Place the chip in the hybridization oven at 65°C. Rotate 5 min at 60 rpm.
Remove the wash buffer and repeat step 6 twice.
Fill with 250 μl storage buffer. Scan chip to check stripping efficiency.
Scan the chip to check the scanned image.
If no signal is detected on the image, proceed to hybridize with the target (re-probe).
Change the oven temperature to 45°C.
Replace the storage buffer with pre-hybridization buffer. Place the chip in the hybridization oven at 45°C. Rotate 5 min at 60 rpm.
If not re-probed immediately, the chip can be stored at 4°C overnight.

1 hour

The use of fresh probes for re-probing is suggested.

If the scanned image is not clean, repeat the stripping step 3 for another 10 min.

The correlation coefficient between the first hybridization (without stripping) and re-hybridization (stripped) should be above 0.9. The range we have is around 0.96.

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

Biotechnology

Biochemistry

Stripping protocol for Affymetrix tiling gene chips

Hoi-Hung Cheung, Wai-Yee Chan, Tin-Lap Lee

Hoi-Hung Cheung

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Wai-Yee Chan

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Tin-Lap Lee

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

DOI:

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Introduction

Reagents

Equipment

Procedure

Timing

Critical Steps

Troubleshooting

Anticipated Results

Comments (4)

Kate McInnerney

commented on 06 April, 2009

commented on 30 June, 2011

Kate McInnerney

commented on 06 April, 2009

commented on 30 June, 2011

Version History

CURRENT STATUS: Posted

Version 1

Metrics

Comments: 4

HTML Views: ...

Subject Areas

Biotechnology

Biochemistry

Method Article

Biotechnology

Biochemistry

Stripping protocol for Affymetrix tiling gene chips

Hoi-Hung Cheung, Wai-Yee Chan, Tin-Lap Lee

Hoi-Hung Cheung

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Wai-Yee Chan

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

Tin-Lap Lee

Section on Developmental Genomics, Laboratory of Clinical Genomics, and Divsion of Information Technology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

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License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Declarations

Introduction

Reagents

Equipment

Procedure

Timing

Critical Steps

Troubleshooting

Anticipated Results

Comments (4)

Kate McInnerney

commented on 06 April, 2009