Post on 11-Jul-2018
Nucleic Acid Research Group 2007-2008 Study:
“A Comparison of Different Priming Strategies for cDNA Synthesis by Reverse Transcriptase as Measured by Real-Time
RT-qPCR”
Nucleic Acid Research Group 2007-2008 Study:
“A Comparison of Different Priming Strategies for cDNA Synthesis by Reverse Transcriptase as Measured by Real-Time
RT-qPCR”
Reverse Transcription Primer Used
0 10 20 30 40 50 60
Random primers
Oligo(dT)
Gene-specific primer
Random primers and oligo(dT) mixed
Sample is provided
# of respondents
20072004
Reverse Transcription Primer Used
0 10 20 30 40 50 60
Random primers
Oligo(dT)
Gene-specific primer
Random primers and oligo(dT) mixed
Sample is provided
# of respondents
20072004
NARG Survey Results for Types of Primers Used for RT Reactions
NARG Survey Results for Types of Primers Used for RT Reactions
Stangegaard, et. al., BioTechniques 40:649, 2006
•Random Pentadecamer (15-mer)
•Assessed resultant cDNA over a micorarray
Stangegaard, et. al., BioTechniques 40:649, 2006
•Random Pentadecamer (15-mer)
•Assessed resultant cDNA over a micorarray
Literature Suggested Long Randomers Were More Effective Than Short Randomers In Generating cDNA
Literature Suggested Long Randomers Were More Effective Than Short Randomers In Generating cDNA
To evaluate priming strategies used in the reverse transcriptase (RT) reaction to make cDNA for use in real-time qPCR
•Compare randomers, oligo(dT), anchored oligo(dT), and GSP
•Compare randomers of different lengths
•Compare combinations of randomers and Oligo (dT)
To evaluate priming strategies used in the reverse transcriptase (RT) reaction to make cDNA for use in real-time qPCR
•Compare randomers, oligo(dT), anchored oligo(dT), and GSP
•Compare randomers of different lengths
•Compare combinations of randomers and Oligo (dT)
Study GoalStudy Goal
Reverse Transcriptase MethodReverse Transcriptase MethodPrimers (synthesized by Integrated DNA Technologies)
Randomers: 6-mer, 9-mer, 12-mer, 15-mer, 18-mer, and 21-merOligo(dT)20 and Anchored Oligo(dT)20All Randomer with oligo (dT) or anchored oligo (dT) combinationsGene Specific Primers: β-Actin, β-Glucuronidase, TATA Binding ProteinNo Primer and No Primer, No RT
RNAFirstChoice® Human Brain Reference RNA (Ambion)One of the same reference RNAs used as an External RNA Control (ERC) in
the MAQC study (Nature Biotechnology: 24, 1132 – 1139, 2006)RT Assay
SuperScript III cDNA synthesis kit per manufacturer’s (Invitrogen Corp) suggested protocol
100 ng total RNA/reactionPrimer (final conc. for 20 μL rxn)
Randomer: 4 μMOligo (dT): 2.5 μMAnchored oligo (dT): 2.5 μMGene specific: 100 nMCombos: 2 μM randomer + 2 μM oligo dT (or anchored oligo dT)
cDNA SynthesisRandomers and Randomer combos: 25°C, 10 min then 50°C, 50 minOligo (dT), Anchored Oligo (dT), GSP: 50°C, 50 min
Triplicate reactions
Primers (synthesized by Integrated DNA Technologies)Randomers: 6-mer, 9-mer, 12-mer, 15-mer, 18-mer, and 21-merOligo(dT)20 and Anchored Oligo(dT)20All Randomer with oligo (dT) or anchored oligo (dT) combinationsGene Specific Primers: β-Actin, β-Glucuronidase, TATA Binding ProteinNo Primer and No Primer, No RT
RNAFirstChoice® Human Brain Reference RNA (Ambion)One of the same reference RNAs used as an External RNA Control (ERC) in
the MAQC study (Nature Biotechnology: 24, 1132 – 1139, 2006)RT Assay
SuperScript III cDNA synthesis kit per manufacturer’s (Invitrogen Corp) suggested protocol
100 ng total RNA/reactionPrimer (final conc. for 20 μL rxn)
Randomer: 4 μMOligo (dT): 2.5 μMAnchored oligo (dT): 2.5 μMGene specific: 100 nMCombos: 2 μM randomer + 2 μM oligo dT (or anchored oligo dT)
cDNA SynthesisRandomers and Randomer combos: 25°C, 10 min then 50°C, 50 minOligo (dT), Anchored Oligo (dT), GSP: 50°C, 50 min
Triplicate reactions
Bioanalyzer Analysis of the Study RNABioanalyzer Analysis of the Study RNA
Quantitative PCR MethodsQuantitative PCR Methods
qPCR AssaycDNA: 10 ng RNA equivalents from RT rxn[Primer]: Individual Lab specifiedProbe: Individual Lab specifiedSet Up Method: Individual Lab specifiedChemistry: Individual Lab specifiedReplication: Each RT rxn run in duplicate wells
qPCR Results CollectedCt values ΔCt = |Ctprimer - Ctno primer|
qPCR AssaycDNA: 10 ng RNA equivalents from RT rxn[Primer]: Individual Lab specifiedProbe: Individual Lab specifiedSet Up Method: Individual Lab specifiedChemistry: Individual Lab specifiedReplication: Each RT rxn run in duplicate wells
qPCR Results CollectedCt values ΔCt = |Ctprimer - Ctno primer|
Study DesignStudy DesignHuman Brain Reference RNA used for Microarray Study
100 ng Total RNA per RT ReactionHuman Brain Reference RNA used for Microarray Study
100 ng Total RNA per RT Reaction
25 RT priming conditions to test 3 genes: β-actin, β-glucouronidase, TATA Binding Protein
RT reactions in triplicate
Random primers
(6)
Gene specific primers
(3)
Oligo (dT)(1)
No primer +enzyme
(1)
Anchored Oligo (dT)
(1)
Random primers
+ Anchored (dT) (6)
Random primers
+ Oligo (dT)(6)
No primer, no enzyme
(1)
SuperScript IIISuperScript III
Duplicate PCR reactions on each RT reactionRun with TaqMan® Chemistries
71 bases71 bases
hβ-Actin transcript (NM_001101)
hβ-Actin transcript (NM_001101)
Total length - 1793 basesTotal length - 1793 bases
695 bases695 baseshβ-Actinhβ-Actin5’5’ 3’3’
hβ-Glucuronidase transcript (NM_000181)
hβ-Glucuronidase transcript (NM_000181)
Total length - 2245 basesTotal length - 2245 bases
344 bases344
baseshβ-GUShβ-GUS5’5’ 3’3’
66 bases
66 bases
hTATA Binding Protein transcript(NM_003194)
hTATA Binding Protein transcript(NM_003194)
Total length - 1867 basesTotal length - 1867 bases
746 bases746 baseshTBPhTBP5’5’ 3’3’
80 bases
80 bases
Real-Time qPCR Assay MapsReal-Time qPCR Assay Maps
Statistical AnalysisStatistical Analysis
The effect of each variable on Ct or ΔCt levels were assessed using a one-way analysis of variance (ANOVA) with the JMP v 5.01 Statistical Discovery Software (SAS Institute, Cary, NC).
The green diamonds represent the mean and the standard error which is a pooled estimate of the variance.
A Student’s t-test was used to assess for significant difference levels (P < 0.05) between the groups contained within each variable.
The effect of each variable on Ct or ΔCt levels were assessed using a one-way analysis of variance (ANOVA) with the JMP v 5.01 Statistical Discovery Software (SAS Institute, Cary, NC).
The green diamonds represent the mean and the standard error which is a pooled estimate of the variance.
A Student’s t-test was used to assess for significant difference levels (P < 0.05) between the groups contained within each variable.
ALL GENES (ASSAYS)ALL GENES (ASSAYS)
Ct
20
30
4012
mer
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
GU
S
NoP
rimer
Olig
o(dT
)
TBP
b-A
ctin
Primer
Ct
20
30
4012
mer
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
GU
S
NoP
rimer
Olig
o(dT
)
TBP
b-A
ctin
Primer
Effect of Primer on Ct (All Genes)Effect of Primer on Ct (All Genes)
P < 0.0001P < 0.0001
Primer Mean Ct (N)NoPrimer A 29.12 (101)
TBP A B 28.99 (33)21mer B C 27.14 (102)GUS B C 26.92 (27)
15mer C 26.78 (102)18mer C 26.66 (101)
15mer/Oligo(dT) C 26.42 (100)12mer C 26.39 (102)
18mer/Oligo(dT) C 26.35 (100)Oligo(dT) C 26.34 (102)
15mer/AnchOligo(dT) C 26.33 (102)AnchOligo(dT) C 26.32 (99)
18mer/AnchOligo(dT) C 26.27 (101)21mer/AnchOligo(dT) C 26.23 (102)
21mer/Oligo(dT) C 26.16 (102)12mer/Oligo(dT) C 26.15 (102)
12mer/AnchOligo(dT) C 26.14 (102)6mer C 26.04 (102)
6mer/AnchOligo(dT) C 26.01 (101)9mer/AnchOligo(dT) C 25.99 (101)
9mer C 25.94 (101)6mer/Oligo(dT) C 25.94 (102)9mer/Oligo(dT) C 25.91 (101)
β-Actin D 20.05 (36)
Level*
*Levels not connected by the same letter are significantly different (p<0.05)
Effect of Primer on Ct (All Genes)Effect of Primer on Ct (All Genes)
delta
Ct
-4
-3
-2
-1
0
1
2
3
4
5
6
7
812
mer
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
GU
S
NoP
rimer
Olig
o(dT
)
TBP
b-A
ctin
Primer
delta
Ct
-4
-3
-2
-1
0
1
2
3
4
5
6
7
812
mer
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
GU
S
NoP
rimer
Olig
o(dT
)
TBP
b-A
ctin
Primer
Effect of Primer on ΔCt (All Genes)Effect of Primer on ΔCt (All Genes)
P < 0.0001P < 0.0001
Primer Mean ΔCt (N)TBP A 4.95 (33) GUS A 4.56 (27)
9mer/Oligo(dT) B 3.45 (101) 9mer B 3.44 (101)
9mer/AnchOligo(dT) B 3.39 (101) 6mer/Oligo(dT) B C 3.37 (102)
6mer B C D 3.28 (102) 6mer/AnchOligo(dT) B C D 3.25 (101)
12mer/Oligo(dT) B C D 3.16 (102) 21mer/Oligo(dT) B C D 3.15 (102)
12mer/AnchOligo(dT) B C D 3.12 (102) 18mer/AnchOligo(dT) B C D E 3.10 (101)
18mer/Oligo(dT) B C D E 3.09 (100) 21mer/AnchOligo(dT) B C D E 3.08 (102) 15mer/AnchOligo(dT) C D E 2.98 (102)
Oligo(dT) C D E 2.97 (102) 12mer D E F 2.92 (102)
AnchOligo(dT) D E F 2.90 (99) 15mer/Oligo(dT) D E F 2.90 (100)
18mer E F G 2.71 (101) 15mer F G H 2.53 (102) β-Actin G H 2.19 (36) 21mer H 2.17 (102)
NoPrimer I 0.11 (101)
Level*Effect of Primer on ΔCt (All Genes)Effect of Primer on ΔCt (All Genes)
*Levels not connected by the same letter are significantly different (p<0.05)*Levels not connected by the same letter are significantly different (p<0.05)
Ct
20
30
40
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
Ct
20
30
40
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
Primer Class Mean (N)
Rand A 26.49 (610)
O(dT) A B 26.34 (102)aO(dT) A B 26.32 (99)
Rand/aO(dT) A B 26.16 (608)
Rand/O(dT) A B 26.15 (607)GSP B 25.06 (96)
Level*
Effect of Primer Classification on CtEffect of Primer Classification on Ct
*Levels not connected by the same letter are significantly different (p<0.05).
P = 0.2238P = 0.2238
delta
Ct
-4
-3
-2
-1
0
1
2
3
4
5
6
7
8
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
delta
Ct
-4
-3
-2
-1
0
1
2
3
4
5
6
7
8
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
Primer Class Mean (N)
GSP A 3.81 (96)Rand/O(dT) B 3.19 (607)
Rand/aO(dT) B 3.15 (608)
O(dT) B C 2.97 (102)
aO(dT) B C 2.90 (99)
Rand C 2.83 (610)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Primer Classification on ΔCtEffect of Primer Classification on ΔCt
P < 0.0001P < 0.0001
RTRep. Level* Mean (N)
A A 26.42 (737)B A 26.39 (746)C A 26.24 (740)
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Reverse Transcriptase Replicate on Ct
Effect of Reverse Transcriptase Replicate on Ct
Ct
20
30
40
A B C
RT Replicate
P = 0.7626P = 0.7626
delta
Ct
-4
-2
0
2
4
6
8
A B C
RT Replicate
delta
Ct
-4
-2
0
2
4
6
8
A B C
RT Replicate
P = 0.4703P = 0.4703
RT Replicate Level* Mean (N)
C A 2.99 (740)A A 2.95 (740)B A 2.89 (746)
Effect of Reverse Transcriptase Replicate on ΔCt
Effect of Reverse Transcriptase Replicate on ΔCt
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
qPCRRep. Level* Mean (N)
1 A 26.4 (1,111)
2 A 26.3 (1,112)
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of qPCR Replicate on CtEffect of qPCR Replicate on CtC
t
20
30
40
1 2
qPCR Replicate
Ct
20
30
40
1 2
qPCR Replicate
P = 0.7098P = 0.7098
delta
Ct
-4
-2
0
2
4
6
8
1 2
qPCR Replicate
delta
Ct
-4
-2
0
2
4
6
8
1 2
qPCR Replicate
P = 0.6326P = 0.6326
qPCRReplicate Level* Mean (N)
2 A 2.96 (1,111)1 A 2.93 (1,112)
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of qPCR Replicate on ΔCtEffect of qPCR Replicate on ΔCt
Ct
20
30
40
Manual Robot
Set up Method
Ct
20
30
40
Manual Robot
Set up Method
Set UpMethod Mean (N)Manual A 27.30 (1,307)
Robot B 25.01 (916)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Set Up Method on CtEffect of Set Up Method on Ct
P < 0.0001P < 0.0001
delta
Ct
-4
-2
0
2
4
6
8
Manual Robot
Set up Method
delta
Ct
-4
-2
0
2
4
6
8
Manual Robot
Set up Method
Set Up Method Mean(N)Manual A 3.01 (1307)Robot B 2.86 (917)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Set Up Method on ΔCtEffect of Set Up Method on ΔCt
P < 0.0287P < 0.0287
Ct
20
30
40
Lab A Lab B Lab C Lab D Lab E Lab F Lab G
Site
Ct
20
30
40
Lab A Lab B Lab C Lab D Lab E Lab F Lab G
Site
P < 0.0001P < 0.0001
Lab Mean (N)Lab A A 29.33 (132)Lab D A 28.59 (388)Lab E B 26.88 (391)Lab G B 26.81 (396)Lab F C 25.77 (396)Lab B D 24.27 (396)Lab C E 21.61 (124)
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Lab on Ct Effect of Lab on Ct
Level*
Ct
20
30
40
Lab B Lab D Lab E Lab F Lab G
Site (All Assays)
Ct
20
30
40
Lab B Lab D Lab E Lab F Lab G
Site (All Assays)
P < 0.0001P < 0.0001
Lab Mean (N)Lab D A 28.59 (388)Lab E B 26.88 (391)Lab G B 26.81 (396)Lab F C 25.77 (396)Lab B D 24.27 (396)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
For Labs that Performed All Assays-Effect of Lab on Ct
For Labs that Performed All Assays-Effect of Lab on Ct
delta
Ct
-4-3-2-1012345678
Lab B Lab D Lab E Lab F Lab G
Site (All Assays)
delta
Ct
-4-3-2-1012345678
Lab B Lab D Lab E Lab F Lab G
Site (All Assays)
P < 0.0001P < 0.0001
Lab Mean (N)Lab B A 4.40 (396)Lab E B 3.14 (391)Lab D B C 3.03 (388)Lab F C 2.88 (396)Lab G D 1.81 (396)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
For Labs that Performed All Assays-Effect of Lab on ΔCt
For Labs that Performed All Assays-Effect of Lab on ΔCt
Ct
20
30
40
GUS TBP b-Actin
Gene
Ct
20
30
40
GUS TBP b-Actin
Gene
P < 0.0001P < 0.0001
Gene (Assay) Mean (N)
TBP A 30.90 (787)GUS B 28.41 (653)
β-Actin C 20.07 (773)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Gene (Assay) on CtEffect of Gene (Assay) on Ct
delta
Ct
-4
-2
0
2
4
6
8
GUS TBP b-Actin
Gene
delta
Ct
-4
-2
0
2
4
6
8
GUS TBP b-Actin
Gene
P < 0.0001P < 0.0001
Gene (Assay) Mean (N)
GUS A 3.58 (653)TBP B 3.20 (787)
β-Actin C 2.16 (773)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Gene (Assay) on ΔCtEffect of Gene (Assay) on ΔCt
General ResultsGeneral Results
•There was a great variation in Ct and ΔCt between labs, though within lab variation between replicates was negligible
• The effect of RT biological replicates and qPCRreplicates was not significant.
•The robotic set up gave significantly lower Ct and higher ΔCt values than manual set-up
•Assay Type (Gene) significantly affected Ct and ΔCt levels. The effects of primer or primer classification on Ct and ΔCt will be evaluated within each assay
•There was a great variation in Ct and ΔCt between labs, though within lab variation between replicates was negligible
• The effect of RT biological replicates and qPCRreplicates was not significant.
•The robotic set up gave significantly lower Ct and higher ΔCt values than manual set-up
•Assay Type (Gene) significantly affected Ct and ΔCt levels. The effects of primer or primer classification on Ct and ΔCt will be evaluated within each assay
β-actinβ-actin
hβ-Actin transcripthβ-Actin transcript
Total length - 1793 basesTotal length - 1793 bases
695 bases695 baseshβ-Actinhβ-Actin5’5’ 3’3’
71 bases71 bases 7 Stem Structuresby m-fold
7 Stem Structuresby m-fold
Randomers, Oligo(dT), aOligo(dT),Rand + O(dT), Rand + aO(dT)
β-Actin
No Primer
No Primer, No RT
Amplification Plot for β-ActinAmplification Plot for β-Actin
Ct
18
19
20
21
22
23
24
25
26
12m
er
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
Olig
o(dT
)
b-A
ctin
Primer Type
P = 0.3768P = 0.3768
Effect of Primer by Assay (β-actin) on CtEffect of Primer by Assay (β-actin) on Ct
Primer Mean21mer A 20.35 (36)
15mer/Oligo(dT) A B 20.24 (35)18mer A B 20.24 (35)
Oligo(dT) A B 20.15 (36)AnchOligo(dT) A B 20.15 (35)
12mer/AnchOligo(dT) A B C 20.12 (36)15mer A B C 20.08 (36)β-Actin A B C 20.05 (36)
21mer/Oligo(dT) A B C 20.03 (36)18mer/Oligo(dT) A B C 20.01 (34)12mer/Oligo(dT) A B C 19.91 (36)
18mer/AnchOligo(dT) A B C 19.90 (35)21mer/AnchOligo(dT) A B C 19.90 (36)15mer/AnchOligo(dT) A B C 19.89 (36)
6mer/Oligo(dT) A B C 19.87 (36)12mer B C 19.84 (36)
6mer/AnchOligo(dT) B C 19.80 (36)9mer/Oligo(dT) B C 19.77 (35)
6mer B C 19.77 (36)9mer/AnchOligo(dT) B C 19.74 (35)
9mer C 19.61 (35)
Level*
Effect of Primer by Assay (β-actin) on CtEffect of Primer by Assay (β-actin) on Ct
*Levels not connected by the same letter are significantly different (p<0.05)*Levels not connected by the same letter are significantly different (p<0.05)
Ct
18
19
20
21
22
23
24
25
26
12m
er
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
NoP
rimer
Olig
o(dT
)
b-A
ctin
Primer
Ct
18
19
20
21
22
23
24
25
26
12m
er
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
NoP
rimer
Olig
o(dT
)
b-A
ctin
Primer
P < 0.0001P < 0.0001
Effect of Primer by Assay (β-actin) on CtEffect of Primer by Assay (β-actin) on Ct
Level Mean (N)NoPrimer A 22.13 (36)
21mer B 20.35 (36)15mer/Oligo(dT) B C 20.24 (35)
18mer B C 20.24 (35)Oligo(dT) B C 20.15 (36)
AnchOligo(dT) B C 20.15 (35)12mer/AnchOligo(dT) B C D 20.12 (36)
15mer B C D 20.08 (36)β-Actin B C D 20.05 (36)
21mer/Oligo(dT) B C D 20.03 (36)18mer/Oligo(dT) B C D 20.01 (34)12mer/Oligo(dT) B C D 19.91 (36)
18mer/AnchOligo(dT) B C D 19.90 (35)21mer/AnchOligo(dT) B C D 19.90 (36)15mer/AnchOligo(dT) B C D 19.89 (36)
6mer/Oligo(dT) B C D 19.87 (36)12mer C D 19.84 (36)
6mer/AnchOligo(dT) C D 19.80 (36)9mer/Oligo(dT) C D 19.77 (35)
6mer C D 19.77 (36)9mer/AnchOligo(dT) C D 19.74 (35)
9mer D 19.61 (35)
Effect of Primer by Assay (β-actin) on CtEffect of Primer by Assay (β-actin) on Ct
*Levels not connected by the same letter are significantly different (p<0.05)*Levels not connected by the same letter are significantly different (p<0.05)
P = 0.6815P = 0.6815
Primer Class Level* Mean (N)O(dT) A 20.15 (36)
aO(dT) A 20.15 (35)GSP (β-actin) A 20.05 (36)
Rand A 19.98 (214)Rand/O(dT) A 19.97 (212)
Rand/aO(dT) A 19.89 (214)
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Ct
18
19
20
21
22
23
24
25
26
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
Effect of Primer Classification by Assay (β-actin) on Ct
Effect of Primer Classification by Assay (β-actin) on Ct
delta
Ct
-4
-3
-2
-1
0
1
2
3
4
5
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
delta
Ct
-4
-3
-2
-1
0
1
2
3
4
5
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
P = 0.7669P = 0.7669
Primer Class Level* Mean (N)Rand/aO(dT) A 2.34 (214)Rand/O(dT) A 2.26 (212)
Rand A 2.25 (214)GSP (β-actin) A 2.19 (36)
O(dT) A 2.09 (36)aO(dT) A 2.07 (35)
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Primer Classification by Assay (β-actin) on ΔCt
Effect of Primer Classification by Assay (β-actin) on ΔCt
β-Glucuronidase (GUS)β-Glucuronidase (GUS)
hβ-Glucuronidase transcripthβ-Glucuronidase transcript
Total length - 2245 basesTotal length - 2245 bases
344 bases344
baseshβ-GUShβ-GUS5’5’ 3’3’
66 bases
66 bases
3 Stem Structuresby m-fold
3 Stem Structuresby m-fold
GUS, Oligo(dT), aOligo(dT),Rand + O(dT), Rand + aO(dT)
Randomers
No Primer
No Primer, No RT
Amplification Plot for GUSAmplification Plot for GUS
Ct
30
40
12m
er
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
GU
S
NoP
rimer
Olig
o(dT
)
Primer
Effect of Primer by Assay (GUS) on Ct Effect of Primer by Assay (GUS) on Ct
P < 0.0001P < 0.0001
Primer Mean (N)NoPrimer A 31.81 (30)
21mer B 30.25 (30)15mer B C 29.99 (30)12mer B C D 29.44 (30)6mer B C D E 29.04 (30)18mer C D E F 28.81 (30)9mer C D E F 28.67 (30)
15mer/AnchOligo(dT) D E F G 28.19 (30)15mer/Oligo(dT) D E F G 28.17 (29)12mer/Oligo(dT) E F G 28.09 (30)
6mer/AnchOligo(dT) E F G 28.06 (29)18mer/Oligo(dT) E F G 27.96 (30)
21mer/AnchOligo(dT) E F G 27.96 (30)
9mer/Oligo(dT) E F G 27.82 (30)18mer/AnchOligo(dT) E F G 27.81 (30)
6mer/Oligo(dT) E F G 27.79 (30)12mer/AnchOligo(dT) F G 27.68 (29)9mer/AnchOligo(dT) F G 27.63 (30)
21mer/Oligo(dT) F G 27.59 (30)AnchOligo(dT) F G 27.55 (29)
Oligo(dT) F G 27.51 (30)GUS G 26.92 (27)
Level*
Effect of Primer by Assay (GUS) on Ct Effect of Primer by Assay (GUS) on Ct
*Levels not connected by the same letter are significantly different (p<0.05)*Levels not connected by the same letter are significantly different (p<0.05)
delta
Ct
-3
-2
-1
0
1
2
3
4
5
6
7
12m
er
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
GU
S
NoP
rimer
Olig
o(dT
)
Primer
delta
Ct
-3
-2
-1
0
1
2
3
4
5
6
7
12m
er
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
GU
S
NoP
rimer
Olig
o(dT
)
Primer
P < 0.0001P < 0.0001
Effect of Primer by Assay (GUS) on ΔCt Effect of Primer by Assay (GUS) on ΔCt
Primer Mean (N)GUS A 4.59 (27)
Oligo(dT) A 4.52 (30)21mer/Oligo(dT) A B 4.43 (30)
9mer/AnchOligo(dT) A B 4.41 (29)
AnchOligo(dT) A B 4.32 (30)12mer/AnchOligo(dT
)A B 4.29 (29)
6mer/Oligo(dT) A B 4.25 (30)18mer/AnchOligo(dT
)A B 4.22 (30)
9mer/Oligo(dT) A B 4.22 (30)21mer/AnchOligo(dT
)A B C 4.08 (30)
18mer/Oligo(dT) A B C 4.07 (30)12mer/Oligo(dT) A B C D 3.94 (30)
6mer/AnchOligo(dT) A B C D 3.92 (29)
15mer/AnchOligo(dT)
A B C D 3.86 (30)
15mer/Oligo(dT) B C D E 3.70 (29)9mer C D E F 3.36 (30)18mer D E F 3.22 (30)6mer E F 2.99 (30)12mer F G 2.59 (30)15mer G 2.04 (30)21mer G 1.79 (30)
NoPrimer H 0.22 (30)
Effect of Primer by Assay (GUS) on ΔCt Effect of Primer by Assay (GUS) on ΔCt Level*
*Levels not connected by the same letter are significantly different (p<0.05)*Levels not connected by the same letter are significantly different (p<0.05)
Ct
30
40
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
Ct
30
40
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
Primer Class Mean (N)Rand A 29.37 (180)
Rand/O(dT) B 27.91 (179)Rand/aO(dT) B 27.89 (178)
aO(dT) B 27.56 (29)O(dT) B 27.52 (30)
GSP (GUS) B 26.93 (27)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Primer Classification by Assay (GUS) on Ct
Effect of Primer Classification by Assay (GUS) on Ct
P < 0.0001P < 0.0001
delta
Ct
-3
-2
-1
0
1
2
3
4
5
6
7
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
delta
Ct
-3
-2
-1
0
1
2
3
4
5
6
7
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
P < 0.0001P < 0.0001
Primer Class Mean (N)GSP (GUS) A 4.59 (27)
O(dT) A 4.52 (30)aO(dT) A 4.32 (29)
Rand/aO(dT) A 4.13 (178)Rand/O(dT) A 4.10 (179)
Rand B 2.67 (180)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Primer Classification By Assay (GUS) on ΔCt
Effect of Primer Classification By Assay (GUS) on ΔCt
TBPTBP
hTATA Binding Protein transcripthTATA Binding Protein transcript
Total length - 1867 basesTotal length - 1867 bases
746 bases746 baseshTBPhTBP5’5’ 3’3’
80 bases
80 bases
8 Stem Structuresby m-fold
8 Stem Structuresby m-fold
Randomers, Oligo(dT), aOligo(dT),Rand + O(dT), Rand + aO(dT)
TBP
No Primer
Amplification Plot for TBPAmplification Plot for TBP
Ct
30
40
12m
er
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
NoP
rimer
Olig
o(dT
)
TBP
Primer
Ct
30
40
12m
er
12m
er/A
nchO
ligo(
dT)
12m
er/O
ligo(
dT)
15m
er
15m
er/A
nchO
ligo(
dT)
15m
er/O
ligo(
dT)
18m
er
18m
er/A
nchO
ligo(
dT)
18m
er/O
ligo(
dT)
21m
er
21m
er/A
nchO
ligo(
dT)
21m
er/O
ligo(
dT)
6mer
6mer
/Anc
hOlig
o(dT
)
6mer
/Olig
o(dT
)
9mer
9mer
/Anc
hOlig
o(dT
)
9mer
/Olig
o(dT
)
Anc
hOlig
o(dT
)
NoP
rimer
Olig
o(dT
)
TBP
Primer
Effect of Primer by Assay (TBP) on CtEffect of Primer by Assay (TBP) on Ct
P < 0.0001P < 0.0001
Primer Mean (N)NoPrimer A 34.02 (35)Oligo(dT) B 31.56 (36)
AnchOligo(dT) B C 31.48 (35)21mer B C D 31.36 (36)
15mer/AnchOligo(dT) B C D E 31.21 (36)18mer/AnchOligo(dT) B C D E F 31.17 (36)
18mer B C D E F 31.13 (36)21mer/AnchOligo(dT) B C D E F 31.13 (36)
21mer/Oligo(dT) B C D E F 31.10 (36)15mer/Oligo(dT) B C D E F 31.02 (36)18mer/Oligo(dT) B C D E F 31.00 (36)
12mer/AnchOligo(dT) B C D E F 30.93 (36)15mer B C D E F 30.81 (36)
12mer/Oligo(dT) B C D E F 30.76 (36)9mer/AnchOligo(dT) B C D E F G 30.69 (36)6mer/AnchOligo(dT) C D E F G 30.57 (36)
6mer/Oligo(dT) D E F G 30.48 (36)12mer E F G 30.40 (36)
9mer/Oligo(dT) F G 30.30 (36)9mer G H 29.82 (36)6mer G H 29.80 (36)TBP H 28.99 (33)
Effect of Primer by Assay (TBP) on CtEffect of Primer by Assay (TBP) on Ct
*Levels not connected by the same letter are significantly different (p<0.05)*Levels not connected by the same letter are significantly different (p<0.05)
Level*
Ct
30
40
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
Ct
30
40
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
P < 0.0001P < 0.0001
Primer Class Mean (N)O(dT) A 31.55 (36)
aO(dT) A B 31.48 (35)
Rand/aO(dT) A B 30.95 (216)Rand/O(dT) B C 30.78 (216)
Rand C 30.55 (216)
GSP (TBP) D 28.99 (33)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Primer Classification by Assay (TBP) on Ct
Effect of Primer Classification by Assay (TBP) on Ct
delta
Ct
-3
-2
-1
0
1
2
3
4
5
6
7
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
delta
Ct
-3
-2
-1
0
1
2
3
4
5
6
7
GSP O(dT) Rand Rand/O(dT) Rand/aO(dT) aO(dT)
Primer Classification
P < 0.0001P < 0.0001
Primer Class Mean (N)GSP (TBP) A 4.95 (33)
Rand B 3.56 (216)Rand/O(dT) C 3.34 (216)
Rand/aO(dT) C 3.16 (216)O(dT) D 2.57 (36)
aO(dT) D 2.55 (35)
Level*
*Levels not connected by the same letter are significantly different (p<0.05).
*Levels not connected by the same letter are significantly different (p<0.05).
Effect of Primer Classification by Assay (TBP) on ΔCt
Effect of Primer Classification by Assay (TBP) on ΔCt
Results-By AssayResults-By Assayβ-Actin
There were not any significant differences between any of the primers or combinations in their ability to affect Ct or ΔCt levels
GUSThe effects of oligo (dT) and anchored oligo (dT) alone or in combination with a randomer on Ct or ΔCt were significant compared to the randomers alone
TBPThe effects of the gene specific primer, followed by the randomers alone or in combination on Ct or ΔCt were significant compared to the effects of oligo (dT) and anchored oligo (dT)
β-ActinThere were not any significant differences between any of the primers or combinations in their ability to affect Ct or ΔCt levels
GUSThe effects of oligo (dT) and anchored oligo (dT) alone or in combination with a randomer on Ct or ΔCt were significant compared to the randomers alone
TBPThe effects of the gene specific primer, followed by the randomers alone or in combination on Ct or ΔCt were significant compared to the effects of oligo (dT) and anchored oligo (dT)
LOCATION?LOCATION?
LOCATION?LOCATION?
LOCATION?LOCATION?
Real-Time qPCR Assay MapsReal-Time qPCR Assay Maps
hβ-Actin transcripthβ-Actin transcript
Total length - 1793 basesTotal length - 1793 bases
695 bases695 baseshβ-Actinhβ-Actin5’5’ 3’3’
71 bases71 bases 7 Stem Structuresby m-fold
7 Stem Structuresby m-fold
hβ-Glucuronidase transcripthβ-Glucuronidase transcript
Total length - 2245 basesTotal length - 2245 bases
344 bases344
baseshβ-GUShβ-GUS5’5’ 3’3’
66 bases
66 bases
3 Stem Structuresby m-fold
3 Stem Structuresby m-fold
hTATA Binding Protein transcripthTATA Binding Protein transcript
Total length - 1867 basesTotal length - 1867 bases
746 bases746 baseshTBPhTBP5’5’ 3’3’
80 bases
80 bases
8 Stem Structuresby m-fold
8 Stem Structuresby m-fold
ConclusionsConclusions
•The use of longer randomers (15-, 18-, and 21-mers) did not perform as well as the shorter randomers (6-and 9-mers) with respect to giving lower Ct values and higher ΔCt differences.
•The use of longer randomers (15-, 18-, and 21-mers) did not perform as well as the shorter randomers (6-and 9-mers) with respect to giving lower Ct values and higher ΔCt differences.
•Oligo (dT) or anchored oligo (dT) appear to be more effective primers than randomers when the assays are designed closer to the 3’ end of the transcript.
•Oligo (dT) or anchored oligo (dT) appear to be more effective primers than randomers when the assays are designed closer to the 3’ end of the transcript.
•Self-priming in the RT reaction can result in the generation of cDNA levels similar to those levels achieved when primers are added.
•Self-priming in the RT reaction can result in the generation of cDNA levels similar to those levels achieved when primers are added.
•Oligo (dT) and anchored oligo (dT) appear to be equally as effective in generating cDNA for use in qPCR•Oligo (dT) and anchored oligo (dT) appear to be equally as effective in generating cDNA for use in qPCR
What is the Best Priming Strategy to Use to Generate cDNA for Use in qPCR?
What is the Best Priming Strategy to Use to Generate cDNA for Use in qPCR?
The use of randomer-oligo (dT) or randomer-anchored oligo (dT) combinations in the RT reaction appear to give universally lower Ct values and higher ΔCt differences regardless of the assay location.
If the cDNA will be used for only 1 gene assay, the appropriate gene-specific primer may be a better choice.
The use of randomer-oligo (dT) or randomer-anchored oligo (dT) combinations in the RT reaction appear to give universally lower Ct values and higher ΔCt differences regardless of the assay location.
If the cDNA will be used for only 1 gene assay, the appropriate gene-specific primer may be a better choice.
Nucleic Acids Research GroupNucleic Acids Research Group
Timothy C. Hunter (Co-Chair)
Kevin L. Knudtson (Co-Chair)
Deborah S. Grove
Deborah J. Hollingshead
Gregory L. Shipley
Katia Sol-Church
William L. Taylor
Kathryn S. Lilley (EB liason)
Timothy C. Hunter (Co-Chair)
Kevin L. Knudtson (Co-Chair)
Deborah S. Grove
Deborah J. Hollingshead
Gregory L. Shipley
Katia Sol-Church
William L. Taylor
Kathryn S. Lilley (EB liason)
University of Vermont
University of Iowa
Penn State University
University of Pittsburgh
UTHSC- Houston
A. I. duPont Hospital for Children
UTHSC- Memphis
Cambridge University
University of Vermont
University of Iowa
Penn State University
University of Pittsburgh
UTHSC- Houston
A. I. duPont Hospital for Children
UTHSC- Memphis
Cambridge University
http://www.abrf.org/NARG
NARG Study: Part IINARG Study: Part II•Rationale: Allow users to benchmark their in-house RT protocol using provided RNA template and primers.
•Rationale: Allow users to benchmark their in-house RT protocol using provided RNA template and primers.
•Real-time qRT-PCR Community performs the Reverse Transcriptase (RT) Reaction
•Provided•RNA•Primers (6-mer, 9-mer, oligo (dT)20, mixes, GSP•Protocols
•Questionnaire/Survey•Return cDNA to the NARG for qPCR
•Real-time qRT-PCR Community performs the Reverse Transcriptase (RT) Reaction
•Provided•RNA•Primers (6-mer, 9-mer, oligo (dT)20, mixes, GSP•Protocols
•Questionnaire/Survey•Return cDNA to the NARG for qPCR
•Goal: To find an effective primer(s) that will provide optimal cDNA synthesis for use in the broadest range of qPCR assays.
•Goal: To find an effective primer(s) that will provide optimal cDNA synthesis for use in the broadest range of qPCR assays.