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