ALL KITS AREN’T CREATED EQUAL!
Comparing cDNA Synthesis Kits
By Ryan Matson
Purpose: Why Compare?
Our last evaluation was a long time ago!
New kits become available
– New primers, modified enzymes, buffers, etc.
– Claim to be more sensitive, efficient, cheaper, etc.
Old kits change
– Suppliers can change ingredients
– Quote expires, cost goes up
Want to ensure GREAT results
Who Cares?
The Genome Core Does!
We do the protocol every day
Work with a wide variety of samples and conditions
We buy lots
We want GREAT results
You Do!
Know what is in your kit, or at least which kit you use
Consistency between projects is key
You care about your results
Evaluating: What to Consider?
Efficiency: Does the kit perform well with
different RNA inputs & with different genes?
Sensitivity: Will the kit transcribe low copy
number transcripts?
Applicability: Can the protocol be incorporated
into the workflow?
Cost: Can we afford it?
Evaluating: Efficiency
RT Linearity - is the kit
efficient with different
inputs of RNA?
Methods
1) Serially dilute RNA
2) Generate cDNA from each
dilution
3) qPCR on cDNA
Evaluating: Efficiency
Methods cont.
– 4) Graph log(CT) vs.
RNA input
– 5) Plug slope into
efficiency equation
Efficiency = 10^(-1/slope)-1
Core’s Acceptable Limits:
0.9 < Efficiency < 1.1
Evaluating: Sensitivity
How well will the kit
transcribe low copy
number transcripts?
Methods
1) Use a standard input of
RNA with all kits
2) qPCR the cDNA
3) Look for amplification &
std deviation
Evaluating: Applicability & Cost
Applicability
–
–
–
–
Ease of use
Time to perform
Number of steps
Number of reagents
Cost
Kits Included for Testing
Applied Biosystems High Capacity cDNA Reverse
Transcription Kit
BioRad iScript cDNA Synthesis Kit
Invitrogen Superscript III First-Strand Synthesis
Supermix
Quanta qScript cDNA Synthesis Kit
Quanta qScript cDNA Supermix
Qiagen Quantitect Reverse Transcription Kit
Stratagene AffinityScript QPCR cDNA Synthesis Kit
Results: Efficiency
Kits varied in their ability to efficiently
generate cDNA from different RNA inputs
Linearity of cDNA Synthesis Kits with b-Actin
40
ABI
35
Average
Invitrogen
iScript
30
Qiagen
Quanta
25
Stratagene
20
15
-1.5
)Supermix (Quanta
-1
-0.5
0
0.5
Log of Dilution
1
1.5
2
2.5
Results: Efficiency
H.Gus
ZEB1
40
40
ABI
35
Qiagen
Quanta
25
Average
Average
iScript
30
ABI
35
Invitrogen
Invitrogen
iScript
30
Qiagen
Quanta
25
Stratagene
Stratagene
15
-1.5
-1
-0.5
0
0.5
1
1.5
2
)Supermix (Quanta
20
)Supermix (Quanta
20
15
-1.5
2.5
-1
-0.5
0
0.5
Log of Dilution
SNAIL
1.5
2
2.5
GapDH Sybr
40
24
ABI
35
22
Invitrogen
iScript
30
Qiagen
Quanta
25
Stratagene
)Supermix (Quanta
20
Average Ct Val
Average
1
Log of Dilution
ABI
Invitrogen
20
iScript
18
Qiagen
16
Quanta
Stratagene
14
)Supermix (Quanta
12
15
-1.5
-1
-0.5
0
0.5
Log of Dilution
1
1.5
2
2.5
10
-1.5
-1
-0.5
0
0.5
Log of Dilution
1
1.5
2
2.5
Results: Efficiency
Pass/Fail Efficiencies for each Gene
Gene
ABI Invitrogen iScript Qiagen QuantaStratagene Supermix
ACTB
0
1
0
0
1
1
1
H.Gus
0
1
1
1
1
0
1
ZEB1
1
1
1
1
1
0
1
SNAIL
1
1
0
1
1
0
1
H.GapDH Sybr
0
0
0
1
0
0
0
H.Gus Sybr
0
0
1
0
1
0
0
Total:
2
4
3
4
5
1
4
Mean Eff:
1.23
1.05 0.97
1.14
1.01
1.36
1.09
1=pass, 0=fail
Quanta>Qiagen,Supermix,Invitrogen>iScript>ABI>Stratagene
Results: Sensitivity
(only with CDH1)
Wells Showing Signal with CDH1 at 5ng/rxn
Wells
Wells
Kit
Amplified
Undermined
ABI
Quanta
Iscript
Qiagen
Invitrogen
15
4
11
13
11
1
12
5
3
5
% Wells
Amplified
0.94
0.25
0.69
0.81
0.69
ABI has most wells amplified and lowest std deviation
(data not shown).
Results: Applicability & Cost
Factors Affecting Protocol
–
–
–
–
# reagent tubes
# of master mixes
# incubation steps
Total incubation time
Result:
Supermix>Quanta,iScript>Stratagene>Invitrogen,ABI>Qiagen
Cost
– Not yet considered
Conclusions
All kits effectively generated cDNA at the
Core’s standard 5ng input
Quanta’s cDNA Sythensis Kit was most
efficient with our test genes
Applied Biosystems kit performed best
with the low copy transcript CDH1
Thanks!
Thanks to:
The Genome Core
– Randy Davis
– Kirsten Copren
– Jenny Dang
– Langdon Smythe
Markus Lacher
The Vendors
– Applied Biosystems
– BioRad
– Invitrogen
– Qiagen
– Quanta
– Stratagene
All of our Users