Hybridization and data processing experiments
Hybridization and data processing experiments
By determining the ratio of fluorescence at each immobilized cDNA spot, one can measure the relative amount of messengers in one sample library to another. If a series of samples are compared to a common reference, the relative amounts of information in all the samples can be compared, yielding similarities and differences between them. Source: Compact Molecular Biology Laboratory Guide, Fifth Edition
Operation method
Hybridization and data processing
Materials and Instruments
Cy3- and Cy5-labeled cDNA glass microarrays Move For details of "Materials", "Reagents" and "Consumables" required for the experiment, see "Others". 1. Calculate the volume of hybridization solution required. As a rule of thumb, 0.033 ul of Hybridization Fluid is required for each square millimeter of slide area covered by a coverslip. A 24 mm x 55 coverslip requires 40 ul of hybridization solution. The amount of hybridization solution added should be sufficient to produce a flat support surface free of air bubbles, so that the coverslip is at the same distance from the slide. 2. 2. For each 40-unit hybridization system, add Cy3- and Cy5-labeled cDNAs to a 0.2 ml thin-walled PCR tube and add DEPC-treated water or remove some water with a Speedvac evaporator to make a volume of 30 ul. 3. Prepare the hybridization system for each of the 40 fractions as listed below: 4. Blow to mix all components, heat on a thermal cycler at 98°C for 2 min, rapidly cool to 25°C and add 0.6 ul 10% SDS. 5. Centrifuge at 16,000 g for 5 min at 20-25°C to allow the coagulated fraction to precipitate. 6. Carefully add the labeled cDNA to a 24 mm x 50 mm coverslip and place it in contact with the inverted downward facing chip. 7. Place the slide into a microarray hybridization cassette. If there is a pool in the cassette, add 5 ml of 3×SSC to the pool; if there is no pool, add to the labeled end of the slide. Seal the hybridization box. Immerse the cassette in a 65°C water bath for 16-20 h. 8. 8. Take the box out of the water bath, cool it and wipe it dry carefully so that moisture from the outside does not get inside when the box is opened. Open the hybridization box and remove the slides. 9. 9. Place the slide with the attached coverslip into a Coplin cylinder filled with 0.5×SSC/0.01% SDS wash. At this point the coverslip will fall off the slide and be picked up with tweezers. Wash the slide in the cylinder for 2 to 5 min. 10. 10. Transfer the slide to another Coplin cylinder filled with 0.06×SSC and wash for 2-5 min. Depending on the source of the RNA sample, the washing procedure may need to be adjusted to remove noise more efficiently. It is helpful to wash the first insert with 0.5×SSC/0.1% SDS or to repeat the routine first step of the wash. 11. 11. Transfer the slide to a slide holder and centrifuge at low speed (167 g or 700-1000 r/min) for 3 min in a clinical centrifuge with a horizontal rotor that can centrifuge the culture plate. Depending on the model and manufacturer used, the details of adjusting the microarray equipment can vary considerably. Similarly, many methods can be used to identify signals in the image, adjust background levels, and normalize values in both channels. This section is simply intended to give some principles for overall consideration in developing images. 12. Adjust the voltage of the photomultiplier and the power of the laser so that the strongest signals are only slightly below the upper limit of the reading [65535 if on a 16-bit scale] and the background values (the rows of dots) are consistently slightly above 0. Then collect the values for the entire image. 13. 13. Enter the captured image into the data extraction software. Review the overall quality of the hybridization: note the consistency and background levels, the signal levels and distribution, the relative strengths of the signals in each channel, and how comparable most of the gene signals are. Caveat All solutions are prepared in RNAase-free water (e.g., DEPC-treated water) unless otherwise noted. Common Problems 1. Materials Glass microarrays Cy3- and Cy5-labeled cDNAs 2. Reagents DEPC-treated water 8 mg/ml poly(dA) 40-60 (Amersham Pharmacia Biotech) 4 mg/ml yeast tRNA 10 mg/ml human C0t-1 DNA 20×SSC 50 x Denhardt solution 10% (m/V) SDS 0.5 × SSC/0.01 % SDS Wash Buffer 0.06 × SSC wash buffer 3. Consumables 0.2 ml thin-walled PCR tubes Thermal cycler 24 mm × 50 mm glass coverslip Microarray hybridization cassette 65°C water bath Microarray Scanner Image Analysis Software For more product details, please visit Aladdin Scientific website.
DEPC-treated water SDS SSC Yeast tRNA Denhardt solution
Thermal cycler 65°C water bath Microarray hybridization cassette Image analysis software
There will be some differences between the chips and the samples, and it will sometimes be necessary to make appropriate adjustments to the hybridization solution fractions. If there is residual hybridization signal from the duplicate DNA control on the chip, it may be appropriate to add more C0t-1 DNA, e.g., high sample closure. If there is a diffuse background on the chip or blurring occurs on all chip elements, add more non-specific containment components, such as high array containment.