Stripping Protocol for Affymetrix Tiling Gene Chips.
Lab/Group: Chan Lab
Introduction
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.
Materials
Reagents
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)
Equipment
Scanner: GeneChip® Scanner 3000 7G.
Fluidics: GeneChip® Fluidics Station 450.
Hybridization Oven: GeneChip® Hybridization Oven 645.
Procedure
1. Pre-warm the stripping buffer at 65°C.
2. Pre-warm the hybridization oven at 65°C.
2. Fill the tiling chip with 200 μl pre-warmed stripping buffer.
3. Place the chip in hybridization oven at 65°C. Rotate 20 min at 60 rpm.
4. Remove stripping buffer. Strip again with another 200 μl of stripping buffer for 10 min.
5. Repeat step 4 for one more time.
6. 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.
7. Remove the wash buffer and repeat step 6 twice.
8. Fill with 250 μl storage buffer. Scan chip to check stripping efficiency.
9. Scan the chip to check the scanned image.
10. If no signal is detected on the image, proceed to hybridize with the target (re-probe).
11. Change the oven temperature to 45°C.
12. Replace the storage buffer with pre-hybridization buffer. Place the chip in the hybridization oven at 45°C. Rotate 5 min at 60 rpm.
13. If not re-probed immediately, the chip can be stored at 4°C overnight.
Troubleshooting
If the scanned image is not clean, repeat the stripping step 3 for another 10 min.
Critical Steps
The use of fresh probes for re-probing is suggested.
Anticipated Results
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.
References
Acknowledgements
Keywords
Tiling microarray, stripping, Gene chip
Comments
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.
Posted by: Kate McInnerney | April 7, 2009 03:59 PM