To determine whether several hybridizations of replicates performed better than hybridization of pooled replicates, we performed two distinct reverse transcription reactions with our sample template and three independent RAP-PCR reactions for each reverse transcription, yielding six PCR probes (Fig.
We next wanted to determine whether the transcriptome coverage of the RAP-PCR products compared with that of whole cDNA.
The distribution of intensities of RAP-PCR and cDNA hybridizations precluded the definition of an objective threshold.
An additional theoretical advantage of using RAP-PCR products is the possibility of having multiple controls if the tag gene is represented in independent RAP-PCBs.
5, as assessed by RAP-PCR hybridization, were confirmed by qRT-PCR (Fig.
The hybridization of RAP-PCR to filter arrays has been shown to solve, in part, these limitations (1).
In the present study, we performed several self-self hybridizations, using distinct conditions, and created a mathematical model to assess the sources of artifactual variability in RAP-PCR hybridization to glass arrays.
We do not know whether the magnitude of artifactual variability resulting from use of RAP-PCR products as a probe compares with that resulting from use of cDNA probes.
Thus, based on our observations, we finally propose performing two hybridizations of distinct pools of RAP-PCR reactions, which is readily amenable for analysis of small samples, either experimental or clinical, increasing the feasibility of using RAP-PCR in the routine clinical setting.
Another potential advantage of hybridization of RAP-PCR probes is the generation of data that may not be easily obtained by conventional cDNA or oligonucleotide-based microarrays because of nonstoichiometric selection of the represented transcripts.
In the experimental samples used to validate our protocol, all changes in expression detected by RAP-PCR array hybridization with log ratios >1.