TABLE 3.
Validation of microarray measurements using real-time RT-PCRa
| Clone ID | Hyb condition | Microarray (fold change) | RT-PCR (fold change) | Putative ID (organism) | E-value |
|---|---|---|---|---|---|
| [1] 13-1 | 713/155 | 5.04 | 53.8 (6.6) | Positive control; Cryphonectria parasitica gene | |
| Euro7/155 | ND | 1.5 (0.1) | |||
| 713/Euro7 | 2.84 | 35.87 | |||
| [2] AEST-02-A-07 (AEST-22-B-11)b | 713/155 | ND | 4.3 (1.1) | SAHH (Saccharomyces cerevisiae) | 7.00E-50 |
| Euro7/155 | 2.35 | 2.6 (0.17) | |||
| 713/Euro7 | ND | 1.65 | |||
| [3] AEST-02-B-01 | 713/155 | ND | 1.4 (0.2) | Heterokaryon incompatibility protein (Saccharomyces cerevisiae) | 1.00E-11 |
| Euro7/155 | ND | −1.0 (0.2) | |||
| 713/Euro7 | ND | 1.4 | |||
| [4] AEST-02-B-12 | 713/155 | ND | 1.3 (0.2) | ABC transporter-like protein (Botryotinia fuckeliana) | 1.00E-36 |
| Euro7/155 | ND | −1.0 (0.1) | |||
| 713/Euro7 | ND | 1.3 | |||
| [5] AEST-02-D-01 | 713/155 | ND | 1.4 (0.3) | DNA replication licensing factor (Aspergillus nidulans) | 3.00E-08 |
| Euro7/155 | ND | −1.1 (0.2) | |||
| 713/Euro7 | ND | 1.54 | |||
| [6] AEST-04-C-01 | 713/155 | ND | 2.0 (0.3) | NADH-cytochrome b5 reductase (Saccharomyces cerevisiae) | 4.00E-34 |
| Euro7/155 | ND | −1.6 (0.2) | |||
| 713/Euro7 | ND | 3.2 | |||
| [7] AEST-04-D-04 | 713/155 | −8.98 | −39.9 (9.2) | Unknown | >E-02 |
| Euro7/155 | −5.12 | −8.0 (2.2) | |||
| 713/Euro7 | −2.52 | −4.9 | |||
| [8] AEST-05-A-09 | 713/155 | −4.73 | −5.5 (1.2) | Aspergillopepsin II precursor (Aspergillus niger) | 2.00E-39 |
| Euro7/155 | −2.08 | −3.1 (0.1) | |||
| 713/Euro7 | ND | −1.77 | |||
| [9] AEST-05-C-02 | 713/155 | −6.20 | −16.9 (5.9) | Hydrogenase regulation HoxX (Aquifex aeolicus) | 3.00E-27 |
| Euro7/155 | −2.30 | −7.0 (0.7) | |||
| 713/Euro7 | ND | −2.4 | |||
| [10] AEST-05-D-11 | 713/155 | −6.78 | −891 (297) | Unknown | >E-02 |
| Euro7/155 | −5.62 | −566 (170) | |||
| 713/Euro7 | −9.02 | −1.57 | |||
| [11] AEST-06-B-03 | 713/155 | −6.19 | −62.5 (20.9) | Possible CGI-83 protein (Leishmania major) | 3.00E-30 |
| Euro7/155 | −3.16 | −17.5 (0.9) | |||
| 713/Euro7 | ND | −3.57 | |||
| [12] AEST-08-D-10 | 713/155 | ND | −2.2 (0.4) | Sperm chromatin HMrBNP/H1 (Pseudopleuronectes americanus) | 1.00E-03 |
| Euro7/155 | ND | 3.6 (1.3) | |||
| 713/Euro7 | −2.18 | −7.92 | |||
| [13] AEST-08-F-10 | 713/155 | 23.68 | 4.2 (0.6) | SAMS (Neurospora crassa) | 3.00E-95 |
| Euro7/155 | 5.8 | 2.1 (0.1) | |||
| 713/Euro7 | 2.37 | 2.0 | |||
| [14] AEST-09-C-08 | 713/155 | ND | 1.2 (0.2) | Proteasome regulatory subunit (Schizosaccharomyces pombe) | 1.00E-55 |
| Euro7/155 | −2.72 | −1.3 (0.2) | |||
| 713/Euro7 | ND | 1.56 | |||
| [15] AEST-09-G-11 | 713/155 | −5.73 | −61.7 (21.7) | Unknown | >E-02 |
| Euro7/155 | −2.74 | −17.5 (2.8) | |||
| 713/Euro7 | ND | −3.53 | |||
| [16] AEST-11-B-12 | 713/155 | −2.78 | −3.3 (0.6) | UTP-glucose-1-phosphate uridylyltransferase (Schizosaccharomyces pombe) | 6.00E-61 |
| Euro7/155 | ND | −1.2 (0.0) | |||
| 713/Euro7 | −2.31 | −2.75 | |||
| [17] AEST-11-E-06 | 713/155 | 3.11 | −1.4 (0.2) | Transcriptional regulator (Clostridium tetani E88) | 7.00E-22 |
| Euro7/155 | 2.91 | 2.4 (0.1) | |||
| 713/Euro7 | 1.96 | −3.36 | |||
| [18] AEST-11-F-02 | 713/155 | ND | 1.7 (0.4) | Ubiquitin/S27a fusion protein (Neurospora crassa) | 2.00E-81 |
| Euro7/155 | 1.93 | −1.1 (0.2) | |||
| 713/Euro7 | ND | 1.87 | |||
| [19] AEST-11-F-11 | 713/155 | −4.15 | −4.8 (0.6) | Chain A, α-1,2-mannosidase (Trichoderma reesei) | 6.00E-33 |
| Euro7/155 | ND | −1.4 (0.1) | |||
| 713/Euro7 | −2.55 | −3.43 | |||
| [20] AEST-12-F-11 | 713/155 | ND | 2.2 (0.3) | AP-1-like transcription factor (Neurospora crassa) | 4.00E-37 |
| Euro7/155 | 2.27 | 4.1 (0.9) | |||
| 713/Euro7 | ND | 0.54 | |||
| [21] AEST-12-G-04 | 713/155 | 4.85 | 6.0 (1.5) | GST (Schizosaccharomyces pombe) | 7.00E-39 |
| Euro7/155 | −3.11 | −4.5 (1.5) | |||
| 713/Euro7 | 4.31 | 27 | |||
| [22] AEST-14-H-02 | 713/155 | −3.00 | −2.5 (0.3) | Polyketide synthase (Mycobacterium leprae) | 1.00E-18 |
| Euro7/155 | ND | −2.8 (0.3) | |||
| 713/Euro7 | ND | 0.89 | /PICK> | ||
| [23] AEST-16-G-09 | 713/155 | ND | −1.9 (0.1) | Nuclear matrix protein (Homo sapiens) | 9.00E-03 |
| Euro7/155 | ND | 2.1 (0.3) | |||
| 713/Euro7 | −2.56 | −3.99 | |||
| [24] AEST-17-C-03 | 713/155 | −2.98 | −2.2 (0.3) | Aspartic protease precursor (Botryotinia fuckeliana) | 1.00E-31 |
| Euro7/155 | 2.15 | 2.2 (0.1) | |||
| 713/Euro7 | −3.33 | −4.85 | |||
| [25] AEST-17-H-07 | 713/155 | ND | 1.2 (0.2) | Probable 26S protease subunit (S. cerevisiae) | 2.00E-91 |
| Euro7/155 | ND | −1.4 (0.2) | |||
| 713/Euro7 | ND | 1.68 | |||
| [26] AEST-17-H-10 | 713/155 | 5.36 | 15.1 (5.0) | Isoamyl alcohol oxidase (Aspergillus oryzae) | 3.00E-42 |
| Euro7/155 | −2.02 | −3.9 (0.8) | |||
| 713/Euro7 | 3.27 | 58.89 | |||
| [27] AEST-22-A-11 | 713/155 | 3.76 | 4.6 (0.4) | Stomatin-like protein (Gibberella fujikuroi) | 2.00E-68 |
| Euro7/155 | 1.90 | 2.3 (0.3) | |||
| 713/Euro7 | 2.24 | 2.0 | |||
| [28] AEST-22-B-01 | 713/155 | ND | 1.9 (0.3) | Annexin XIV (Neurospora crassa) | 2.00E-34 |
| Euro7/155 | ND | −1.3 (0.2) | |||
| 713/Euro7 | ND | 2.47 | |||
| [29] AEST-22-D-09 | 713/155 | ND | 1.2 (0.2) | Ubiquitin-activating enzyme E1 (Candida albicans) | 1.00E-47 |
| Euro7/155 | ND | 1.0 (0.2) | |||
| 713/Euro7 | ND | 1.2 | |||
| [30] AEST-22-H-05 | 713/155 | ND | 4.5 (0.3) | Serine/threonine kinase (Rattus norvegicus) | 1.00E-04 |
| Euro7/155 | ND | 1.8 (0.5) | |||
| 713/Euro7 | 2.15 | 2.5 | |||
| [31] AEST-24-D-01 | 713/155 | 2.02 | 2.5 (0.4) | SnodProt1 precursor (Neurospora crassa) | 2.00E-32 |
| Euro7/155 | 2.92 | 2.9 (0.3) | |||
| 713/Euro7 | ND | 0.86 | |||
| [32] AEST-25-B-11 | 713/155 | −3.54 | −5.9 (1.3) | Nuclease P1 (Penicillium citrinum) | 1.00E-75 |
| Euro7/155 | ND | −1.1 (0.1) | |||
| 713/Euro7 | −3.18 | −5.36 | |||
| [33] AEST-26-B-03 | 713/155 | 3.94 | 8.2 (1.0) | Predicted protein (Neurospora crassa) | 1.00E-36 |
| Euro7/155 | −1.81 | 1.2 (0.4) | |||
| 713/Euro7 | 2.63 | 6.83 | |||
| [34] AEST-27-F-10 | 713/155 | −3.2 | −6.3 (0.4) | Pro1 (Neurospora crassa) | 4.00E-31 |
| Euro7/155 | ND | −1.8 (0.2) | |||
| 713/Euro7 | −2.1 | −3.5 | |||
| [35] AEST-27-H-09 | 713/155 | ND | −1.5 (0.3) | Vivid PAS protein (Neurospora crassa) | 1.00E-38 |
| Euro7/155 | ND | 2.1 (0.2) | |||
| 713/Euro7 | −2.53 | −3.15 | |||
| [36] AEST-30-C-09 | 713/155 | −4.95 | −2.4 (0.3) | Mst12 (Magnaporthe grisea) | 6.00E-65 |
| Euro7/155 | ND | −1.47 (0.22) | |||
| 713/Euro7 | −1.97 | −1.63 | |||
| [37] AEST-30-G-04 | 713/155 | 2.94 | 4.5 (0.8) | Sir2-like (Saccharomyces cerevisiae) | 3.00E-27 |
| Euro7/155 | ND | −1.1 (0.2) | |||
| 713/Euro7 | 2.18 | 4.95 | |||
| [38] AEST-31-F-09 | 713/155 | 8.90 | 16.3 (2.0) | Fum12p (Gibberella moniliformis) | 5.00E-35 |
| Euro7/155 | 4.67 | 7.9 (0.5) | |||
| 713/Euro7 | ND | 2.06 | |||
| [39] AEST-32-A-06 | 713/155 | −5.44 | −15.5 (4.2) | MAS3 protein (Magnaporthe grisea) | 4.00E-11 |
| Euro7/155 | ND | −1.6 (0.4) | |||
| 713/Euro7 | −4.15 | −9.69 |
Real-time RT-PCR measurements of 39 clones. Measurements were made in triplicate for each clone by using two independent total RNA preparations. Clone IDs are indicated in column 1. Column 2 indicates the sources of RNA used for hybridization reactions and real-time RT-PCR measurements: 713/155, transcript accumulation in EP155/CHV1-EP713 relative to virus-free strain EP155; Euro7/155, transcript accumulation in EP155/CHV1-Euro7 relative to virus-free strain EP155; and 713/Euro7, transcript accumulation in EP155/CHV1-EP713 relative to EP155/CHV1-Euro7. Column 3 indicates the average change (n-fold) for each clone ID calculated from microarray experiments (six total hybridizations, from three sets of dye-swap experiments). Column 4 indicates the average change (n-fold) for each clone ID as measured by real-time RT-PCR, with the standard error of the mean in parentheses. Column 5 provides a brief biological process description from the BLAST hit provided in column 6. Underscored text indicates false-negative and boldface text indicates false-positive microarray predictions revealed by real-time RT-PCR analysis. Differential expression based on real-time RT-PCR measurements was defined as a change in transcript accumulation of twofold or greater. ND, no change detected by microarray analysis.
The discrepancy between microarray predictions and real-time RT-PCR measurements for AEST-02-A-07 in CHV1-EP713-infected EP155 prompted an examination of related AEST clones on the primary CHV1-EP713 and CHV1-Euro7 responsive gene lists. The gene SAHH (gi 28924052) is represented by four AEST clones on the C. parasitica microarray chip: AEST-22-B-11, AEST-40-D-10, AEST-01-H-09, and AEST-02-A-07. In this case, the gene was not included on the virus common list (Table 1) because none of the four AESTs were found on both the CHV1-EP713 and CHV1-Euro7 primary responsive lists; AEST-22-B-11, AEST-40-D-10, and AEST-01-H-09 were on the CHV1-EP713-responsive list and AEST-02-A-07 was on the CHV1-Euro7-responsive list. AEST-22-B-11, shown in parentheses, is the representative for gi number 28924052 on the CHV1-EP713 published list (1). The reader is referred to the microarray data management section of Materials and Methods for further explanation.