Transcript Profiling of RAW, J774, AtT, and B Cell

1. Transcript Profiling of RAW, J774, AtT, and B Cell • DNA microarray • Affymetrix GeneChip MOE 430 A & B set provides comprehensive coverage of the transcribed mouse genome • Comprised of 45,265 probe sets • Over 39,000 transcripts and variants can be detected. This chip includes 34,000 well substantiated mouse genes • Cell Lines • RNAs were prepared by Cell Lab • RAW 264.7 : macrophage line (duplicate) • J774A.1 : macrophage line (duplicate) • AtT-20 : pituitary line • B Cell

2. Number of Genes Present AtT > RAW > B Cell > J774 Total 45,265 Elements

3. Number of Genes Present by Chromosome Y chromosome test1 test2 test1 test2 X chromosome RAW: male J774: female AtT: female B Cell: male

4. Chemotaxis (in RAW) GenMAPP Red: Present Yellow: Absent

5. Major Differences Remain in Several Different Gene Families J774A.1 B Cell RAW 264.7 AtT-20 PKC Rho

6. Expression of Receptors J774 (She) > AtT (She) > RAW (He) > B Cell (He) J774 > AtT > RAW > B Cell

7. Receptors Expressed in RAW and J774 Clues for selection of the initial ligands to test Cytokine Receptors – Hematopoetin Receptor Family test1 test2 test1 test2 Serotonin Receptors

8. Receptors Expressed in RAW and J774 (continued) Adenosine, Adrenoceptors, or Calcitonin Gene-Related Peptide Receptors test1 test2 test1 test2 Other Receptors

9. J774 Chemotaxis RAW C5a SDF1 MCP1 fMLP (Courtesy of JI) Chemokine Receptors test1 test2 test1 test2

10. Identify Receptors That Are Expressed in RAW but NOT in J774 RAW } Probably Right } } There are Pitfalls

11. Identify Receptors That Are Expressed in J774 but NOT in RAW What are the PITFALLS ? J774 ? ? ? _at: Probe designed for anti-sense target _a_at: Designates probe sets that recognize multiple transcripts from the same gene. _s_at: Designates probe sets that share common probes among multiple transcripts from different genes. _x_at: Designates probe sets where it was not possible to select either a unique probe set or a probe set with identical probes among multiple transcripts. Rules for cross-hybridization were dropped. Therefore, these probe sets may cross-hybridize in an unpredictable manner with other sequences.

12. Real Identification of Receptors That Are Expressed Cytokine Receptors (TNF family) _at: Probe designed for anti-sense target _s_at: Designates probe sets that share common probes among multiple transcripts from different genes. _x_at: Designates probe sets where it was not possible to select either a unique probe set or a probe set with identical probes among multiple transcripts. Rules for cross-hybridization were dropped. Therefore, these probe sets may cross-hybridize in an unpredictable manner with other sequences. _a_at: Designates probe sets that recognize multiple transcripts from the same gene.

13. Things to Consider in Chip • The oligo design is an important factor for obtaining accurate transcript levels and there are certainly some non-specific oligos in Affymetrix GeneChip. • There are factors to consider before interpreting the microarray result, such as _a_at (multiple transcripts from the same Gene), _s_at (multiple transcripts from different genes), _x_at (may cross-hybridize in an unpredictable manner with other sequences). • When the level of intensity of the element islow, the Present/Absent call information may be inaccurate.

14. Summaries • RAW and B Cell were males while J774 and AtT were females. • The number of genes present is higher in AtT (about 10%). RAW, J774, and B Cell are similar to each other. • The number of expressed receptor genes is 5-10% higher in J774. • Using Affymetrix chips some of the different gene family members can be distinguished (GenMAPP). • There is a relationship between the expression level of transcript and the related biological function (Chemotaxis). • The presence of receptor transcript can suggest the nature of the ligand that will activate the cell.

15. Acknowledgments • Sun Young Lee • Anna Cao • Becky Hart • Robert Hsueh • Christine Horvath • Ron Taussig • Mel Simon