Nucleic Acid Research Group 2007-2008 Study: “A … bases71 bases h β-Actin transcript ...

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)


344 bases344

baseshβ-GUShβ-GUS5’5’ 3’3’

66 bases

66 bases

hTATA Binding Protein transcript(NM_003194)

hTATA Binding Protein transcript(NM_003194)


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



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



delta

Ct

-4

-3

-2

-1

0

1

2

3

4

5

6

7

8



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*



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)



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



qPCRRep. Level* Mean (N)

1 A 26.4 (1,111)

2 A 26.3 (1,112)



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)



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*



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*



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)



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


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*



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


Site (All Assays)

delta

Ct

-4-3-2-1012345678


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*



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*



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*



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



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*



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


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)



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)



Ct

18

19

20

21

22

23

24

25

26



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



delta

Ct

-4

-3

-2

-1

0

1

2

3

4

5



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)



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


344 bases344


66 bases

66 bases



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


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*


Ct

30

40



Ct

30

40



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*



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



delta

Ct

-3

-2

-1

0

1

2

3

4

5

6

7



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*



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



80 bases

80 bases



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


Level*

Ct

30

40



Ct

30

40



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*



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



delta

Ct

-3

-2

-1

0

1

2

3

4

5

6

7



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*



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



71 bases71 bases 7 Stem Structuresby m-fold


hβ-Glucuronidase transcripthβ-Glucuronidase transcript


344 bases344


66 bases

66 bases



hTATA Binding Protein transcripthTATA Binding Protein transcript



80 bases

80 bases



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.

Nucleic Acid Research Group 2007-2008 Study: “A … bases71 bases h β-Actin transcript ...

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Transcript of Nucleic Acid Research Group 2007-2008 Study: “A … bases71 bases h β-Actin transcript ...