Figure 13

Schematic illustration of the effect of washing of the microarray using two rounds of staining and washing: The different steps are characterized as follows: Hybridization: The grey areas refer to the amount of PM and MM probe oligomers occupied with specific (S) and nonspecific (N) transcripts (see Eq. (1)). Both probe types are assumed to hybridize identically with non-specific transcripts (P-N, P = PM,MM). Free probe oligomers are not shown. 1^ststaining: A certain fraction of bound transcripts becomes 'bright' by labeling with fluorescent markers (SAPE) whereas the remaining non-labeled fraction remains 'dark'. The amount of bright probe duplexes of each type in this first labeling round is set to 100%. 1^stwashing: Washing removes bound targets of the bright fraction from the probes as indicated by the arrows (Eq. (4)). The yield of washing depends on the duplex type: The percentage of reduction of bound targets is largest for nonspecific transcripts and smallest for specific transcripts bound to PM probes. The dark fraction is not affected by washing. 2^ndstaining: We assume that in the second staining round the same amount of dark probes is transferred into bright ones by labeling as in the first round. The given percentages refer to the amount of bright probes relative to the initial level after 1^st staining. For example, the amount of bright PM-S duplexes nearly doubles from 90% to 100%+90% = 190%. 2^ndwashing: The amount of bound targets reduces by the same duplex-specific factor as in the 1^st washing round. For example, 90% of the 190% bright PM-S remain bound (0.9·190% = 171% of the initial level). Note that each staining/washing round enriches high-affinity bright duplexes compared with low-affinity bright ones, e.g. PM-S compared with MM-S and with P-N as indicated in the graph in the upper part of the figure. The given enrichment factors for the expression degree PM-S and the ratios PM-S/MM-S and PM-S/PM-N refer to the second round compared with the first one (see text). Note that the respective hook parameters are related logarithmically to the enrichment factors.