pathway specific cdna arrays

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 min95 °C, 5 min

Step 2: Reverse TranscriptaseReaction

Total RNA 5’ 3’

Gene-Specific Primers (A)

70 °C, 3 min42 °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 min95 °C, 5 min

Step 2: ReverseTranscriptaseReaction

Total RNA 5’ 3’

Gene-Specific primers (A)

70 °C, 3 min42 °C, 2 min

Step 1: Annealing Mixture

TrueLabeling Minimizes Endogenous RNA Priming

Enzyme

Conventional

TrueLabeling

Prim

ers

-

+

-

+

1:2 Serial Dilutions of cDNA probe

Endogenous RNA Priming CausesHigh 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

LPR30 cycles

conventionallabeling

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 min37 °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 min85 °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 3072 °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

3.01.50.750.40.1

30 cycles of LPR

g RNA

ID4

LPR Signal is Proportional to Input Total RNA

0

5000

10000

15000

20000

25000

30000

0 1 2 3

BMPR2

ID4

TCF8

TIMP1

g RNA

back

grou

nd c

orre

cted

sign

al in

tens

ity

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

LPR Signal Is Proportional to Cycle Number

0

10000

20000

30000

40000

50000

60000

10 15 20 25 30

BMPR1AJUNMADH2TGFBR1

cycles

back

grou

nd c

orre

cted

sign

al in

tens

ity

AmpoLabeling-LPR Represents Gene Expression Profiles More Accurately

Human TGF/BMP Signaling Pathway GEArray Q Series (HS-023)Human Universal Reference Total RNA

LPRconventional RT-PCR

ACVR1,2

COL3A1

TGFBR2

ACTB

INHAJUN, MADH2

AmpoLabeling-LPR Represents Gene Expression Profiles More Accurately

Mouse Insulin Signaling Pathway GEArray Q Series (MM-030)Mouse Liver or Thymus RNA

LPR

THYMUSLIVER

RT-PCR

G6PC

AGP-1BPKC

UCP2

LIV

ER

TH

YM

US

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

y = 0.7476x + 0.038 R2 = 0.186

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

-0.5 -0.25 0 0.25 0.5

Log

(Liv

er:T

hym

us)

for

RT

-PC

R

Log (Liver:Thymus) for conventional method

Comparing Relative Gene Expression ProfilesObtained by RT-PCR and the Conventional Method

y = 0.4192x + 0.0449 R2 = 0.6456

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

-1 -0.5 0 0.5 1

Log

(Liv

er:T

hym

us)

for

RT

-PC

R

Log (Liver:Thymus) for AmpoLabeling-LPR

Comparing Relative Gene Expression ProfilesObtained by RT-PCR and AmpoLabeling-LPR

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

ACTBFOS

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

LPRconventional

Acaca Akt3Bcl2l CebpbG6pc

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, 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)

Acknowledgements

• Ying Han• Kun Lu• Xiao Zeng

THANK YOU.

Pathway specific cDNA arrays