To analyze reproducibility and sources of variation, we performed several self-self hybridizations of RAP-PCR products, as described above, labeled with distinct fluorochromes and hybridized to conventional cDNA microarrays (Fig.
RAP-PCR VARIABILITY CAN BE EFFECTIVELY REDUCED THROUGH POOLING
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.
All changes with log ratios higher than 1.5, 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.