Pathway specific cDNA arrays

1. Pathway specific cDNA arrays

2. SuperArray Introduces Labeling Protocols Accessory Products for GEArray Original, Q, and S Series Kits

3. Three New Labeling Kits and Protocols • RT-Labeling Enzyme (RT) L-01 • Conventional reverse transcriptase and RNase inhibitor set • TrueLabeling-RT (TL-RT) L-02 • Reduces high background and “false positive” signals by minimizing endogenous priming of RNA • AmpoLabeling-LPR (LPR) L-03(N) • Amplifies the signal intensity of limiting amounts of total RNAor low abundance messages • Detects message ordinarily missed by conventional methods • Represents expression profile more accurately

4. Three New Labeling Kits and Protocols • RT-Labeling Enzyme (RT) L-01 • Conventional reverse transcriptase and RNase inhibitor set • TrueLabeling-RT (TL-RT) L-02 • Reduces high background and “false positive” signals by minimizing endogenous priming of RNA • AmpoLabeling-LPR (LPR) L-03(N) • Amplifies the signal intensity of limiting amounts of total RNAor low abundance messages • Detects message ordinarily missed by conventional methods • Represents expression profile more accurately

5. Gene-Specific Primers (A) 70 °C, 3 min 42 °C, 2 min Step 1: Annealing Mixture 5’ 3’ Total RNA RNase Inhibitor (RI) Reverse Transcriptase (RE) 42 °C, 25 or 90 min 95 °C, 5 min Step 2: Reverse Transcriptase Reaction 5’ 3’ Total RNA 3’ 5’ probe How Does the RT-Labeling Enzyme Protocol Work?

6. Advantages of RT-Labeling Enzyme • Experiments optimized previously using GEArray Original or Q Series Kits • Least expensive

7. Three New Labeling Kits and Protocols • RT-Labeling Enzyme (RT) L-01 • Conventional reverse transcriptase and RNase inhibitor set • TrueLabeling-RT (TL-RT) L-02 • Reduces high background and “false positive” signals by minimizing endogenous priming of RNA • AmpoLabeling-LPR (LPR) L-03(N) • Amplifies the signal intensity of limiting amounts of total RNAor low abundance messages • Detects message ordinarily missed by conventional methods • Represents expression profile more accurately

8. 5’ 3’ Total RNA Gene-Specific primers (A) 70 °C, 3 min 42 °C, 2 min Step 1: Annealing Mixture 5’ 3’ Total RNA RNase Inhibitor (RI) TrueLabeling Reverse Transcriptase (AE) 42 °C, 90 min 95 °C, 5 min Step 2: Reverse Transcriptase Reaction 3’ 5’ probe How does the TrueLabeling-RT Enzyme Protocol Work?

9. Primers Enzyme - Conventional + - TrueLabeling + TrueLabeling Minimizes Endogenous RNA Priming 1:2 Serial Dilutions of cDNA probe

10. Gene-specific primers - + Conventional TrueLabeling Endogenous RNA Priming Causes High Background and a Skewed Expression Profile Human TGFb/BMP Signaling Pathway GEArray Q Series (HS-023) Human Universal Reference Total RNA

11. Advantages of TrueLabeling-RT • Minimizes endogenous RNA priming • Lowers background • Reduces possible false positive signals

12. Three New Labeling Kits and Protocols • RT-Labeling Enzyme (RT) L-01 • Conventional reverse transcriptase and RNase inhibitor set • TrueLabeling-RT (TL-RT) L-02 • Reduces high background and “false positive” signals by minimizing endogenous priming of RNA • AmpoLabeling-LPR (LPR) L-03(N) • Amplifies the signal intensity of limiting amounts of total RNAor low abundance messages • Detects message ordinarily missed by conventional methods • Represents expression profile more accurately

13. VS conventional labeling LPR 30 cycles AmpoLabeling-LPR Human TGFb/BMP Signaling Pathway GEArray Q Series (HS-023) 3.0 mg Human Universal Reference Total RNA

14. 5’ 3’ Total RNA Random Primers (P) 70 °C, 3 min 37 °C, 10 min 5’ 3’ Total RNA How Does the AmpoLabeling-LPR Labeling Protocol Work? Step 1: Annealing Mixture +

15. 5’ 3’ mRNA RNase Inhibitor (RI) Reverse Transcriptase (RE) 37 °C, 25 min 85 °C, 5 min 3’ 5’ cDNA RE How Does the AmpoLabeling-LPR Labeling Protocol Work? Step 2: RT Reaction

16. 3’ 5’ cDNA Gene-specific primers (AF) Thermo-stable DNA Polymerase (LE) 85 °C, 5 min (85 °C, 1 min; 50 °C, 1 min., 72 °C, 1 min.) x 30 72 °C, 5 min 5’ 3’ probe How Does the AmpoLabeling-LPR Labeling Protocol Work? Step 3: Linear Polymerase Replication

17. 30 cycles of LPR ID4 mg RNA 0.75 3.0 1.5 0.1 0.4 LPR Signal Is Proportional to Input Total RNA:As Little as 0.1 mg of Total RNA Detectable Human TGFb/BMP Signaling Pathway GEArray Q Series (HS-023) Human Universal Reference Total RNA

18. background corrected signal intensity mg RNA LPR Signal is Proportional to Input Total RNA

19. 20 25 10 30 15 JUN cycles of LPR LPR Signal Is Proportional to Cycle Number:No More Than 30 Cycles are Required Human TGFb/BMP Signaling Pathway GEArray Q Series (HS-023) 3.0 mg Human Universal Reference Total RNA

20. LPR Signal Is Proportional to Cycle Number background corrected signal intensity cycles

21. RT-PCR conventional LPR ACVR1,2 COL3A1 INHA JUN, MADH2 TGFBR2 ACTB AmpoLabeling-LPR Represents Gene Expression Profiles More Accurately Human TGFb/BMP Signaling Pathway GEArray Q Series (HS-023) Human Universal Reference Total RNA

22. RT-PCR LPR G6PC AGP-1B PKCb UCP2 LIVER THYMUS LIVER THYMUS AmpoLabeling-LPR Represents Gene Expression Profiles More Accurately Mouse Insulin Signaling Pathway GEArray Q Series (MM-030) Mouse Liver or Thymus RNA

23. conventional LPR RT-PCR LIVER THYMUS AmpoLabeling-LPR Represents Gene Expression Profiles More Accurately Mouse Insulin Signaling Pathway GEArray Q Series (MM-030) Mouse Liver or Thymus RNA

24. Log (Liver:Thymus) for RT-PCR Log (Liver:Thymus) for conventional method Comparing Relative Gene Expression ProfilesObtained by RT-PCR and the Conventional Method

25. Log (Liver:Thymus) for RT-PCR Log (Liver:Thymus) for AmpoLabeling-LPR Comparing Relative Gene Expression ProfilesObtained by RT-PCR and AmpoLabeling-LPR

26. conventional 30 cycles LPR AKT1 FOS ACTB VS PDK1 LPR Detects Low Abundance Messages Ordinarily Missed by Conventional Methods GEArray Original Series Human PI-3 Kinase & AKT (hGEA9912030) 7.5 mg Human Universal Reference Total RNA Chemiluminescent Detection

27. conventional LPR Acaca Akt3 Bcl2l Cebpb G6pc VS Pdpk1 LPR Detects Low Abundance Messages Ordinarily Missed by Conventional Methods GEArray Q Series Mouse Insulin Signaling Pathway (MM-030) Mouse Universal Reference Total RNA Radioactive Detection

28. AmpoLabeling-LPR conventional VS LPR Detects Low Abundance Messages Ordinarily Missed by Conventional Methods Luo, Y., Cai, J., Ginis, I., Rao, M. (2003) Manuscript in Preparation GEArray S Series Mouse Stem Cell (MM-601.1) 0.6 mg Mouse D3 ES cell and 0.2 mg adult rat hippocampus total RNA Chemiluminescent Detection

29. Advantages of AmpoLabeling-LPR • Detects lower abundance messages • Detects message ordinarily missed by conventional methods • Represents gene expression profile more accurately • Use smaller amounts of input total RNA • Minimizes problems due to endogenous RNA priming • Lowers background • Reduces possible false positive (and false negative) signals

30. Three New Labeling Kits and Protocols • RT-Labeling Enzyme (RT) L-01 $ 50 • Includes: RI, RE, B, BN, C, D, E, H2O • TrueLabeling-RT (TL-RT) L-02 $ 100 • Includes: RI, AE, B, BN, C, D, E, H2O • AmpoLabeling-LPR (LPR) L-03(N) $ 200 • Includes: P, RI, RE, L, LE, BL, BN, C, H2O)

31. Acknowledgements • Ying Han • Kun Lu • Xiao Zeng THANK YOU.

32. Pathway specific cDNA arrays