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Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines).

Klink VP, Overall CC, Alkharouf NW, Macdonald MH, Matthews BF - J. Biomed. Biotechnol. (2010)

Bottom Line: The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods.Conclusion.DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Harned Hall, Mississippi State University, Mississippi State, MS 39762, USA.

ABSTRACT
Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process.

No MeSH data available.


Detection calls.  (a, c, e, g, i)  Gene counts per sample.  (b, d,  f,  h, j)  Functional categories for the histograms.   P: pericycle; I3: incompatible syncytium-3 dpi; C3: compatible syncytium 3 dpi; C8: compatible syncytium-8 dpi; DE: differentially expressed. The functional categories are as follows: A: Cell Growth & Division; B: Cell Structure; C: Disease & Defense; D: Energy; E: Intracellular Traffic; F: Metabolism; G: No Homology to Known Proteins; H: Post-Transcription; I: Protein Destination & Storage; J: Protein Synthesis; K: Secondary Metabolism; L: Signal Transduction; M: Transcription; N: Transporter; O: Transposon; P: Unclassified-Hypothetical Protein NOT Supported by cDNA; Q: Unclassified-Hypothetical Protein Supported by cDNA; R: Unclassified-Protein with Unknown Function.  (a) Venn diagram depicting P versus I3. The three portions of the Venn diagram show transcripts for  P-present-unique; common-present; I3-present-unique.  (b)  Functional categorization depicting P versus I3. The seven histograms (per functional category) are  P-present-unique; common-present; I3-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26]  and those that were present: I3-present & DE; I3-DE-induced; I3 dpi-DE-suppressed; I3-present & not DE.  (c) Venn diagram depicting P versus C3. The three portions of the Venn diagram show transcripts for  P-present-unique; common-present; C3-present-unique.  (d)  Functional categorization depicting P versus C3. The seven histograms (per functional category) are  P-present-unique; common-present; C3-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C3-present & DE; C3-DE-induced; C3-DE-suppressed; C3-present & not DE.  (e) Venn diagram depicting P versus C8. The three portions of the Venn diagram show transcripts for  P-present-unique; common-present; C8 present-unique.  (f)  Functional categorization depicting P versus C8. The seven histograms (per functional category) are  P-present-unique; common-present; C8-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C8-present & DE; C8-DE-induced; C8-DE-suppressed; C8-present & not DE.  (g)  Venn diagram depicting C3 versus I3. The three portions of the Venn diagram show transcripts for  C3-present-unique; common-present; I3-present-unique.  (h)  Functional categorization depicting C3 versus I3. The eight histograms (per functional category) are  C3-present-unique; common-present; I3-present-unique. The remaining five transcript identifications represent comparisons between those that measured DE [26] and those that were present: I3-present & DE; I3-DE-induced; I3-DE-suppressed; I3-present & not DE; C3-present & not DE.  (i)  Venn diagram depicting C3 versus C8. The three portions of the Venn diagram show transcripts for  C3-present-unique; common-present; C8-present-unique.  (j)  Functional categorization depicting C3 versus C8. The seven histograms (per functional category) are  C3-present-unique; common-present; C8-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C8-present & DE; C8-DE-induced; I3-DE-suppressed; C8-present & not DE.
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fig8: Detection calls. (a, c, e, g, i) Gene counts per sample. (b, d, f, h, j) Functional categories for the histograms. P: pericycle; I3: incompatible syncytium-3 dpi; C3: compatible syncytium 3 dpi; C8: compatible syncytium-8 dpi; DE: differentially expressed. The functional categories are as follows: A: Cell Growth & Division; B: Cell Structure; C: Disease & Defense; D: Energy; E: Intracellular Traffic; F: Metabolism; G: No Homology to Known Proteins; H: Post-Transcription; I: Protein Destination & Storage; J: Protein Synthesis; K: Secondary Metabolism; L: Signal Transduction; M: Transcription; N: Transporter; O: Transposon; P: Unclassified-Hypothetical Protein NOT Supported by cDNA; Q: Unclassified-Hypothetical Protein Supported by cDNA; R: Unclassified-Protein with Unknown Function. (a) Venn diagram depicting P versus I3. The three portions of the Venn diagram show transcripts for P-present-unique; common-present; I3-present-unique. (b) Functional categorization depicting P versus I3. The seven histograms (per functional category) are P-present-unique; common-present; I3-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: I3-present & DE; I3-DE-induced; I3 dpi-DE-suppressed; I3-present & not DE. (c) Venn diagram depicting P versus C3. The three portions of the Venn diagram show transcripts for P-present-unique; common-present; C3-present-unique. (d) Functional categorization depicting P versus C3. The seven histograms (per functional category) are P-present-unique; common-present; C3-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C3-present & DE; C3-DE-induced; C3-DE-suppressed; C3-present & not DE. (e) Venn diagram depicting P versus C8. The three portions of the Venn diagram show transcripts for P-present-unique; common-present; C8 present-unique. (f) Functional categorization depicting P versus C8. The seven histograms (per functional category) are P-present-unique; common-present; C8-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C8-present & DE; C8-DE-induced; C8-DE-suppressed; C8-present & not DE. (g) Venn diagram depicting C3 versus I3. The three portions of the Venn diagram show transcripts for C3-present-unique; common-present; I3-present-unique. (h) Functional categorization depicting C3 versus I3. The eight histograms (per functional category) are C3-present-unique; common-present; I3-present-unique. The remaining five transcript identifications represent comparisons between those that measured DE [26] and those that were present: I3-present & DE; I3-DE-induced; I3-DE-suppressed; I3-present & not DE; C3-present & not DE. (i) Venn diagram depicting C3 versus C8. The three portions of the Venn diagram show transcripts for C3-present-unique; common-present; C8-present-unique. (j) Functional categorization depicting C3 versus C8. The seven histograms (per functional category) are C3-present-unique; common-present; C8-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C8-present & DE; C8-DE-induced; I3-DE-suppressed; C8-present & not DE.

Mentions: Comparisons were made between the different sample types (e.g., 3 dpi incompatible syncytia versus pericycle). In those comparisons, four arrays would be compared (Figure 5). Probe sets that measured detectable amounts of a transcript on the four arrays under comparison were considered common and present between two sample types (e.g., pericycle and 3 dpi incompatible syncytia-common). The probe sets measuring absent on the four arrays (common and absent), although potentially interesting, were not taken into consideration in this analysis. The samples whose probe sets measured present for both arrays but only in one of the two sample types would be considered present and unique for a particular sample type (e.g., pericycle-unique or 3 dpi incompatible syncytia-unique) (Figure 5). Probe sets that measure detectable amounts of gene activity in both sample types can either be differentially expressed or not differentially expressed (Figure 6). The differential expression calls used in some of the comparative analyses had been presented previously [26]. The DCM analysis presented here is employed as a different way of examining the data with the goal of identifying genes at low thresholds of expression that are missed in differential expression analyses. More importantly, DCM is also a way of identifying genes that may be expressed at high thresholds in one sample type and are undetectable in a second sample type used for comparative purposes in a differential expression analysis. In cases like these, statistical analyses cannot be done because no expression data is available for the second sample type and thus the probe set is excluded from the differential expression analysis. Therefore, probe sets that measured detectable amounts of a transcript uniquely in one sample type (e.g., unique-present) (Figure 5) cannot measure differential expression (Figure 6). An example of genes identified in a comparative analysis of two hypothetical gene pools (Figure 7) illustrates the different gene categories investigated in the analysis (Figure 8). As illustrated, all genes that are identified as differentially expressed had to be present in each gene pool (Figure 7). It became clear from the analysis that many genes that were unique to a specific sample type (e.g., A or B) were being excluded from the differential expression analysis because the probe sets measured detectable levels of gene activity only in one of the two sample types (Figure 7). The probe sets that match this criterion, A or B and not A U B, became the focus of the analysis (Figure 8).


Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines).

Klink VP, Overall CC, Alkharouf NW, Macdonald MH, Matthews BF - J. Biomed. Biotechnol. (2010)

Detection calls.  (a, c, e, g, i)  Gene counts per sample.  (b, d,  f,  h, j)  Functional categories for the histograms.   P: pericycle; I3: incompatible syncytium-3 dpi; C3: compatible syncytium 3 dpi; C8: compatible syncytium-8 dpi; DE: differentially expressed. The functional categories are as follows: A: Cell Growth & Division; B: Cell Structure; C: Disease & Defense; D: Energy; E: Intracellular Traffic; F: Metabolism; G: No Homology to Known Proteins; H: Post-Transcription; I: Protein Destination & Storage; J: Protein Synthesis; K: Secondary Metabolism; L: Signal Transduction; M: Transcription; N: Transporter; O: Transposon; P: Unclassified-Hypothetical Protein NOT Supported by cDNA; Q: Unclassified-Hypothetical Protein Supported by cDNA; R: Unclassified-Protein with Unknown Function.  (a) Venn diagram depicting P versus I3. The three portions of the Venn diagram show transcripts for  P-present-unique; common-present; I3-present-unique.  (b)  Functional categorization depicting P versus I3. The seven histograms (per functional category) are  P-present-unique; common-present; I3-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26]  and those that were present: I3-present & DE; I3-DE-induced; I3 dpi-DE-suppressed; I3-present & not DE.  (c) Venn diagram depicting P versus C3. The three portions of the Venn diagram show transcripts for  P-present-unique; common-present; C3-present-unique.  (d)  Functional categorization depicting P versus C3. The seven histograms (per functional category) are  P-present-unique; common-present; C3-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C3-present & DE; C3-DE-induced; C3-DE-suppressed; C3-present & not DE.  (e) Venn diagram depicting P versus C8. The three portions of the Venn diagram show transcripts for  P-present-unique; common-present; C8 present-unique.  (f)  Functional categorization depicting P versus C8. The seven histograms (per functional category) are  P-present-unique; common-present; C8-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C8-present & DE; C8-DE-induced; C8-DE-suppressed; C8-present & not DE.  (g)  Venn diagram depicting C3 versus I3. The three portions of the Venn diagram show transcripts for  C3-present-unique; common-present; I3-present-unique.  (h)  Functional categorization depicting C3 versus I3. The eight histograms (per functional category) are  C3-present-unique; common-present; I3-present-unique. The remaining five transcript identifications represent comparisons between those that measured DE [26] and those that were present: I3-present & DE; I3-DE-induced; I3-DE-suppressed; I3-present & not DE; C3-present & not DE.  (i)  Venn diagram depicting C3 versus C8. The three portions of the Venn diagram show transcripts for  C3-present-unique; common-present; C8-present-unique.  (j)  Functional categorization depicting C3 versus C8. The seven histograms (per functional category) are  C3-present-unique; common-present; C8-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C8-present & DE; C8-DE-induced; I3-DE-suppressed; C8-present & not DE.
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Related In: Results  -  Collection

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fig8: Detection calls. (a, c, e, g, i) Gene counts per sample. (b, d, f, h, j) Functional categories for the histograms. P: pericycle; I3: incompatible syncytium-3 dpi; C3: compatible syncytium 3 dpi; C8: compatible syncytium-8 dpi; DE: differentially expressed. The functional categories are as follows: A: Cell Growth & Division; B: Cell Structure; C: Disease & Defense; D: Energy; E: Intracellular Traffic; F: Metabolism; G: No Homology to Known Proteins; H: Post-Transcription; I: Protein Destination & Storage; J: Protein Synthesis; K: Secondary Metabolism; L: Signal Transduction; M: Transcription; N: Transporter; O: Transposon; P: Unclassified-Hypothetical Protein NOT Supported by cDNA; Q: Unclassified-Hypothetical Protein Supported by cDNA; R: Unclassified-Protein with Unknown Function. (a) Venn diagram depicting P versus I3. The three portions of the Venn diagram show transcripts for P-present-unique; common-present; I3-present-unique. (b) Functional categorization depicting P versus I3. The seven histograms (per functional category) are P-present-unique; common-present; I3-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: I3-present & DE; I3-DE-induced; I3 dpi-DE-suppressed; I3-present & not DE. (c) Venn diagram depicting P versus C3. The three portions of the Venn diagram show transcripts for P-present-unique; common-present; C3-present-unique. (d) Functional categorization depicting P versus C3. The seven histograms (per functional category) are P-present-unique; common-present; C3-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C3-present & DE; C3-DE-induced; C3-DE-suppressed; C3-present & not DE. (e) Venn diagram depicting P versus C8. The three portions of the Venn diagram show transcripts for P-present-unique; common-present; C8 present-unique. (f) Functional categorization depicting P versus C8. The seven histograms (per functional category) are P-present-unique; common-present; C8-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C8-present & DE; C8-DE-induced; C8-DE-suppressed; C8-present & not DE. (g) Venn diagram depicting C3 versus I3. The three portions of the Venn diagram show transcripts for C3-present-unique; common-present; I3-present-unique. (h) Functional categorization depicting C3 versus I3. The eight histograms (per functional category) are C3-present-unique; common-present; I3-present-unique. The remaining five transcript identifications represent comparisons between those that measured DE [26] and those that were present: I3-present & DE; I3-DE-induced; I3-DE-suppressed; I3-present & not DE; C3-present & not DE. (i) Venn diagram depicting C3 versus C8. The three portions of the Venn diagram show transcripts for C3-present-unique; common-present; C8-present-unique. (j) Functional categorization depicting C3 versus C8. The seven histograms (per functional category) are C3-present-unique; common-present; C8-present-unique. The remaining four transcript identifications represent comparisons between those that measured DE [26] and those that were present: C8-present & DE; C8-DE-induced; I3-DE-suppressed; C8-present & not DE.
Mentions: Comparisons were made between the different sample types (e.g., 3 dpi incompatible syncytia versus pericycle). In those comparisons, four arrays would be compared (Figure 5). Probe sets that measured detectable amounts of a transcript on the four arrays under comparison were considered common and present between two sample types (e.g., pericycle and 3 dpi incompatible syncytia-common). The probe sets measuring absent on the four arrays (common and absent), although potentially interesting, were not taken into consideration in this analysis. The samples whose probe sets measured present for both arrays but only in one of the two sample types would be considered present and unique for a particular sample type (e.g., pericycle-unique or 3 dpi incompatible syncytia-unique) (Figure 5). Probe sets that measure detectable amounts of gene activity in both sample types can either be differentially expressed or not differentially expressed (Figure 6). The differential expression calls used in some of the comparative analyses had been presented previously [26]. The DCM analysis presented here is employed as a different way of examining the data with the goal of identifying genes at low thresholds of expression that are missed in differential expression analyses. More importantly, DCM is also a way of identifying genes that may be expressed at high thresholds in one sample type and are undetectable in a second sample type used for comparative purposes in a differential expression analysis. In cases like these, statistical analyses cannot be done because no expression data is available for the second sample type and thus the probe set is excluded from the differential expression analysis. Therefore, probe sets that measured detectable amounts of a transcript uniquely in one sample type (e.g., unique-present) (Figure 5) cannot measure differential expression (Figure 6). An example of genes identified in a comparative analysis of two hypothetical gene pools (Figure 7) illustrates the different gene categories investigated in the analysis (Figure 8). As illustrated, all genes that are identified as differentially expressed had to be present in each gene pool (Figure 7). It became clear from the analysis that many genes that were unique to a specific sample type (e.g., A or B) were being excluded from the differential expression analysis because the probe sets measured detectable levels of gene activity only in one of the two sample types (Figure 7). The probe sets that match this criterion, A or B and not A U B, became the focus of the analysis (Figure 8).

Bottom Line: The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods.Conclusion.DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Harned Hall, Mississippi State University, Mississippi State, MS 39762, USA.

ABSTRACT
Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process.

No MeSH data available.