A problem with cDNA production is that not all mRNA's are reverse-transcribed with the same efficiency. This fact leads to reverse transcription bias, which can change the relative amounts of different cDNA's measured by the microarray assay. Reverse transcription bias is not a problem when comparing the same mRNA across two cell populations unless it causes the mRNA not to be transcribed at all. However, the bias does prohibit quantitative comparison between different mRNA's on one array. Another problem caused by bias is that some mRNA's may be reverse-transcribed for only part of their lengths, making them less likely to bind and stay bound to their complements on the array. One way of getting around this problem is to prime reverse transcription from random starting positions on the mRNA's, rather than always starting from their tails. This method can reduce bias, but it also makes useless cDNA from any remaining ribosomal and transfer RNA's in the sample.

3. Fluorescent labeling of cDNA's

In order to detect cDNA's bound to the microarray, we must label them with a reporter molecule that identifies their presence. The reporters currently used in comparative hybridization to microarrays are fluorescent dyes (fluors), represented by the red and green circles attached to the cDNA's in the diagram [2]. A differently-colored fluor is used for each sample so that we can tell the two samples apart on the array. The labeled cDNA samples are called probes because they are used to probe the collection of spots on the array.

The colors of the fluors in the diagram are just for illusrtration. The actual fluors do not show their colors unless stimulated with a specific frequency of light by a laser. Even then, the colors are not directly observed; rather, the wavelength of the emitted light is used to tune a detector which measures the fluorescence. The choice of red and green colors is reminiscent of the emission wavelengths of commonly-used fluors, such as rhodamine and fluorescein or Cy3 and Cy5.

The number of fluor molecules which label each cDNA depends on its length and possibly its sequence composition, both of which are often unknown. This is one more reason that fluorescent intensities for different cDNA's cannot be quantitatively compared. However, identical cDNA's from the two probes are still comparable as long as the same number of label molecules are added to the same DNA sequence in each probe.

To equalize the total concentrations of the two cDNA probes before applying them to the array, the probe solutions are diluted to have the same overall fluorescent intensity. This procedure makes two possibly unjustified assumptions: first, that the total amount of mRNA in each cell type being tested is identical; and second, that each fluor emits the same amount of light relative to its concentration. The second assumption can be eliminated by suitable calibration, but the first one is difficult to check. It may therefore be that cells with more abundant mRNA are made into a probe with artifically low mRNA concentrations.

4. Hybridization to a DNA Microarray

The two cDNA probes are tested by hybridizing them to a DNA microarray. The array holds hundreds or thousands of spots, each of which contains a different DNA sequence. If a probe contains a cDNA whose sequence is complementary to the DNA on a given spot, that cDNA will hybridize to the spot, where it will be detectable by its fluorescence. In this way, every spot on an array is an independent assay for the presence of a different cDNA. There is enough DNA on each spot that both probes can hybridize to it at once without interference.

Microarrays are made from a collection of purified DNA's. A drop of each type of DNA in solution is placed onto a specially-prepared glass microscope slide by an