pathway specific cdna arrays
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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