April 2008 cDNA Synthesis Kits Evaluationcancer.ucsf.edu/.../cdna_synthesis_kits_eval_0408.pdf ·...
Transcript of April 2008 cDNA Synthesis Kits Evaluationcancer.ucsf.edu/.../cdna_synthesis_kits_eval_0408.pdf ·...
ALL KITS ARENALL KITS AREN’’T CREATED EQUAL!T CREATED EQUAL!Comparing cDNA Synthesis KitsComparing cDNA Synthesis Kits
By Ryan MatsonBy Ryan Matson
Purpose: Why Compare?Purpose: Why Compare?
Our last evaluation was a long time ago!Our last evaluation was a long time ago!New kits become availableNew kits become available–– New primers, modified enzymes, buffers, etc.New primers, modified enzymes, buffers, etc.–– Claim to be more sensitive, efficient, cheaper, etc.Claim to be more sensitive, efficient, cheaper, etc.
Old kits changeOld kits change–– Suppliers can change ingredientsSuppliers can change ingredients–– Quote expires, cost goes upQuote expires, cost goes up
Want to ensure Want to ensure GREATGREAT resultsresults
Who Cares?Who Cares?
The Genome Core Does!The Genome Core Does!We do the protocol every dayWe do the protocol every dayWork with a wide variety of samples and conditionsWork with a wide variety of samples and conditionsWe buy lotsWe buy lotsWe want GREAT resultsWe want GREAT results
You Do!You Do!Know what is in your kit, or at least which kit you useKnow what is in your kit, or at least which kit you useConsistency between projects is keyConsistency between projects is keyYou care about your resultsYou care about your results
Evaluating: What to Consider?Evaluating: What to Consider?
EfficiencyEfficiency: Does the kit perform well with : Does the kit perform well with different RNA inputs & with different genes?different RNA inputs & with different genes?SensitivitySensitivity: Will the kit : Will the kit transcribetranscribe low copy low copy number transcripts?number transcripts?ApplicabilityApplicability: Can the protocol be incorporated : Can the protocol be incorporated into the workflow? into the workflow? CostCost: Can we afford it?: Can we afford it?
Evaluating: EfficiencyEvaluating: EfficiencyRT Linearity RT Linearity -- is the kit is the kit efficient with different efficient with different inputs of RNA?inputs of RNA?MethodsMethods1) Serially dilute RNA1) Serially dilute RNA2) Generate cDNA from each 2) Generate cDNA from each
dilutiondilution3) qPCR on cDNA3) qPCR on cDNA
Evaluating: EfficiencyEvaluating: Efficiency
Methods cont.Methods cont.–– 4) Graph log(CT) vs. 4) Graph log(CT) vs.
RNA inputRNA input–– 5) Plug slope into 5) Plug slope into
efficiency equationefficiency equation
Efficiency = 10^(-1/slope)-1
Core’s Acceptable Limits:0.9 < Efficiency < 1.1
Evaluating: SensitivityEvaluating: SensitivityHow well will the kit How well will the kit transcribetranscribe low copy low copy number transcripts?number transcripts?MethodsMethods1) Use a standard input of 1) Use a standard input of
RNA with all kitsRNA with all kits2) 2) qPCRqPCR the cDNAthe cDNA3) Look for amplification & 3) Look for amplification &
std deviationstd deviation
Evaluating: Applicability & CostEvaluating: Applicability & CostApplicabilityApplicability–– Ease of useEase of use–– Time to performTime to perform–– Number of stepsNumber of steps–– Number of reagentsNumber of reagents
CostCost
Kits Included for TestingKits Included for TestingApplied Applied BiosystemsBiosystems High Capacity cDNA Reverse High Capacity cDNA Reverse Transcription KitTranscription KitBioRadBioRad iScriptiScript cDNA Synthesis KitcDNA Synthesis KitInvitrogen Superscript III FirstInvitrogen Superscript III First--Strand Synthesis Strand Synthesis SupermixSupermixQuanta Quanta qScriptqScript cDNA Synthesis KitcDNA Synthesis KitQuanta Quanta qScriptqScript cDNAcDNA SupermixSupermixQiagenQiagen QuantitectQuantitect Reverse Transcription KitReverse Transcription KitStratagene Stratagene AffinityScriptAffinityScript QPCR cDNA Synthesis KitQPCR cDNA Synthesis Kit
Results: EfficiencyResults: Efficiency
Kits varied in their ability to efficiently Kits varied in their ability to efficiently generate cDNA from different RNA inputsgenerate cDNA from different RNA inputs
Linearity of cDNA Synthesis Kits with b-Actin
15
20
25
30
35
40
-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
Log of Dilution
Ave
rage
ABI
Invitrogen
iScript
Qiagen
Quanta
Stratagene
)Supermix (Quanta
Results: EfficiencyResults: Efficiency
ZEB1
15
20
25
30
35
40
-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
Log of Dilution
Ave
rage
ABI
Invitrogen
iScript
Qiagen
Quanta
Stratagene
)Supermix (Quanta
H.Gus
15
20
25
30
35
40
-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
Log of Dilution
Ave
rage
ABI
Invitrogen
iScript
Qiagen
Quanta
Stratagene
)Supermix (Quanta
SNAIL
15
20
25
30
35
40
-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
Log of Dilution
Ave
rage
ABI
Invitrogen
iScript
Qiagen
Quanta
Stratagene
)Supermix (Quanta
GapDH Sybr
10
12
14
16
18
20
22
24
-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
Log of Dilution
Aver
age
Ct V
alABIInvitrogeniScriptQiagenQuantaStratagene)Supermix (Quanta
Results: EfficiencyResults: Efficiency
Pass/Fail Efficiencies for each GeneGene ABI Invitrogen iScript QiagenQuantaStratagene Supermix ACTB 0 1 0 0 1 1 1H.Gus 0 1 1 1 1 0 1ZEB1 1 1 1 1 1 0 1SNAIL 1 1 0 1 1 0 1H.GapDH Sybr 0 0 0 1 0 0 0H.Gus Sybr 0 0 1 0 1 0 0Total: 2 4 3 4 5 1 4Mean Eff: 1.23 1.05 0.97 1.14 1.01 1.36 1.091=pass, 0=fail
Quanta>Qiagen,Supermix,Invitrogen>iScript>ABI>Stratagene
Results: SensitivityResults: Sensitivity(only with CDH1)(only with CDH1)
Wells Showing Signal with CDH1 at 5ng/rxn
Kit Wells Amplified
Wells Undermined
% Wells Amplified
ABI 15 1 0.94 Quanta 4 12 0.25 Iscript 11 5 0.69 Qiagen 13 3 0.81 Invitrogen 11 5 0.69
ABI has most wells amplified and lowest std deviation(data not shown).
Results: Applicability & CostResults: Applicability & CostFactors Affecting ProtocolFactors Affecting Protocol–– # reagent tubes# reagent tubes–– # of master mixes# of master mixes–– # incubation steps# incubation steps–– Total incubation timeTotal incubation time
Result:Result:SupermixSupermix>>Quanta,iScriptQuanta,iScript>Stratagene>>Stratagene>Invitrogen,ABIInvitrogen,ABI>>QiagenQiagen
CostCost–– Not yet consideredNot yet considered
ConclusionsConclusions
All kits effectively generated cDNA at the All kits effectively generated cDNA at the CoreCore’’s standard 5ng inputs standard 5ng inputQuantaQuanta’’s cDNA s cDNA SythensisSythensis Kit was most Kit was most efficient with our test genesefficient with our test genesApplied Applied BiosystemsBiosystems kit performed best kit performed best with the low copy transcript CDH1with the low copy transcript CDH1
Thanks!Thanks!
Thanks to:Thanks to:The Genome CoreThe Genome Core–– Randy DavisRandy Davis–– Kirsten CoprenKirsten Copren–– Jenny DangJenny Dang–– Langdon SmytheLangdon Smythe
Markus Markus LacherLacher
The VendorsThe Vendors–– Applied Applied BiosystemsBiosystems–– BioRadBioRad–– InvitrogenInvitrogen–– QiagenQiagen–– QuantaQuanta–– StratageneStratagene
All of our UsersAll of our Users