Pathway specific cDNA arrays

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

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

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

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

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

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

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

TrueLabeling Minimizes Endogenous RNA Priming Enzyme Conventional TrueLabeling Primers :2 Serial Dilutions of cDNA probe

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

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

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

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

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

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

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 °C, 5 min Gene-specific primers (AF) 3’5’ cDNA 3’ 5’ probe

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 cycles of LPR  g RNA ID4

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

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 cycles of LPR JUN

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

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

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

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

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

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

GEArray Original Series Human PI-3 Kinase & AKT (hGEA ) 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

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

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

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

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)

Acknowledgements Ying Han Kun Lu Xiao Zeng THANK YOU.

Pathway specific cDNA arrays