Phase 2 Validation and Troubleshooting

While preparing a manuscript for publication, and nearing the end of our array experiments, we undertook a series of experiments aimed at confirming the array data with more "traditional" means of northern blotting and RT-PCR [17]. Confirming array cDNA expression data by other independent means is an important control whenever arrays are being used [18]. We selected six cDNAs that appeared to be highly specific for one or more of the three cell types under study and ordered the corresponding bacterial glycerol stocks from ResGen. Clones were identified by accession numbers and were sequence-verified upon arrival in our lab. Oligonucleotide primers specific to each cDNA were designed for use in PCR so

that the resulting products would only contain the insert sequences from each plasmid. These PCR products were gel purified and used to probe northern blots, and later, for the dot blots described below.

In three out of six cases, Northern blotting failed to confirm our array data. One of the cDNAs did not hybridize to RNA on a northern blot with the same cell type distribution as indicated on the array. Another two did not hybridize to anything at all on a Northern blot, even though the genes appeared to be highly expressed on our arrays (Fig. 7.2). RT-PCR, using the same RNA used for array analysis, was also used in an attempt to confirm the expression results seen with the array experiments. Again, in the same three of the six cases, the expression profile we expected was not observed, calling into question the validity of the array results.

Our initial inquiries with the technical support staff at ResGen gave us no indication of how our data analysis could be flawed. The arrays themselves were said to be highly reliable because of the quality control measures employed by Res-Gen. Additionally, there had been no reports from any of the other customers using these particular arrays that would have suggested any problems with the identity of the spots on the array. After several attempts to validate the clones in question with northern blotting and RT-PCR, we decided to undertake a series of "dot blot" hybridizations to check whether or not the cDNAs that we had selected were indeed present on the gf300 arrays in the grid locations indicated by Res-Gen.

Our technique was to pick a single cDNA in question, generate a clonal probe by PCR-amplifying the insert, gel purifying the product, labeling it with 33P-dCTP, and hybridizing this product to a fresh gf300 array. Our expectation was

Fig. 7.2 A Individual spots representative of hybridization signals seen on the gf300 array corresponding to the location of the Brain Finger Protein (BFP, Accession number AA997188), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, accession number AA924111). These images represent the signals seen on three individual arrays hybridized to cardiomyocyte, cardiac fibroblast, and smooth muscle cell probes. A strong BFP hybridization signal is evident with the cardiomyocyte but not with the fibroblast or smooth muscle probes. B Northern blot with ~20 ^g of total RNA from the indicated samples loaded in each lane hybridized to the BFP probe. GAPDH was included in the hybridization as a control. The ~1.3 kb GAPDH transcript is indicated with an arrow, and the expected location of the BFP transcript at ~3.7 kb is indicated with an arrowhead. De spite the intense signal present at the BFP location on the array when probed with cardi-omyocyte RNA, no significant BFP expression was seen by northern blotting. C Dot blot using a gf300 test microarray hybridized to the same BFP probe that was used on the northern blot shown in panel A. Two high intensity signals are observed on the array after hybridization instead of a single spot at location field 1, grid F, column 6, row 14 expected for the BFP clone. The observed positions of hybridization signal are 1, E, 6, 14 and 1, F, 6, 13. The same result was obtained from similar arrays from three independent printing lots. Upon sequence verification, the clone at position 1, F, 6, 14 was found to be an unidentified EST unrelated to brain finger protein. D Close up of the E—F section of the array showing the location of the two hybridization signals.

that a single clone would produce a single, high intensity hybridization spot on the gf300 array, and furthermore, that the spot would be present in the predicted location on the array grid. Arrays hybridized in this manner were processed according to the standard hybridization and washing protocol for use on a complex probe. This approach, which is similar to a cDNA dot blot, was undertaken on six individual cDNAs. (As an aside, each gf300 array cost 1000.00 and we had no expectation that the filters would be re-usable after such a test. The decision to pursue this line of inquiry thus was not taken lightly.)

One example of our array dot blot results is shown in Fig. 7.2 C. We selected one of the cDNAs whose expression by northern did not match the array result (GenBank accession number AA997188, Brain Finger Protein [19]) and hybridized it to a fresh gf300 array. To our surprise, the result gave two discreet hybridization signals on the array grid (Fig. 7.2), and both were in the wrong grid location. The two other cDNAs that were problematic by northern and RT-PCR were similarly problematic on array dot blots. One of the clones hybridized to a different location than expected, and the other clone did not hybridize to any grid location on the array. The three other cDNA clones whose northern analysis agreed with the original array data appeared in the predicted location as a single intense hybridization signals by array dot blot.

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