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 RNA or 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 RNA or low abundance messages –Detects message ordinarily missed by conventional methods –Represents expression profile more accurately

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

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 RNA or low abundance messages –Detects message ordinarily missed by conventional methods –Represents expression profile more accurately

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

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

10. Endogenous RNA Priming Causes High Background and a Skewed Expression Profile Conventional TrueLabeling -+ Gene-specific primers Human TGF  /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 RNA or low abundance messages –Detects message ordinarily missed by conventional methods –Represents expression profile more accurately

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

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

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

16. How Does the AmpoLabeling-LPR Labeling Protocol Work? Step 3: Linear Polymerase Replication 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 Gene-specific primers (AF) 3’5’ cDNA 3’ 5’ probe

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

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

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

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

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

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

23. LIVER conventional LPR RT-PCR 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 Profiles Obtained by RT-PCR and the Conventional Method

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

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

27. GEArray Q Series Mouse Insulin Signaling Pathway (MM-030) Mouse Universal Reference Total RNA Radioactive Detection LPR Detects Low Abundance Messages Ordinarily Missed by Conventional Methods VS LPR conventional AcacaAkt3 Bcl2lCebpb G6pc Pdpk1

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

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, H 2 O TrueLabeling-RT (TL-RT)L-02$ 100 –Includes: RI, AE, B, BN, C, D, E, H 2 O AmpoLabeling-LPR (LPR)L-03(N)$ 200 –Includes: P, RI, RE, L, LE, BL, BN, C, H 2 O)

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

32. Pathway specific cDNA arrays