DNA/RNAExtraction&Qualification

Date:2009‐04‐07Version:1.0

The Quality Control Platform (QC PF) of Saint‐Louis, funded by the «Ligue nationale contre lecancer», is dedicated to the quality control of biological resources in the framework of the CITprogram(PlatformdirectedbyDrMiraAYADI‐cit.qcplatform@gmail.com).

Process:

A. Collectionoftheconsortiumsamples(tissue,cells,lysate,DNA,RNA,miRNA)B. Extractionofnucleicacids(ifrequired)C. Qualificationofnucleicacidsformicroarraysand/orreal‐timePCRapplications

A. CollectionoftheconsortiumsamplesTheidentificationofthesamplesiscontrolledorperformedaccordingthisformat<projectalias>_<projetcinvestigatoralias>_<speciesalias>_<samplenumber>.Allinformationaboutthesamplesarecollectedinadatabase(databasesoftware:FileMakerPro)toensurethetraceability.Storageconditions:

‐ Tissue,cells,lysate,RNAandmiRNAarestoredat‐80°C‐ DNAarestoredat‐20°CB. Qualificationofnucleicacids

Nucleicacidsareextractedbasedonstandardizedmethods:DNA:

- Manual‐Phenol/Chloroform- Manual‐QIAampDNAMini(Qiagen)

RNA:

- Manual‐Trizol- Manual‐RNeasyMicro(Qiagen)- Manual‐RNeasyMini(Qiagen)

DNA/RNAsimultaneously:

- Manual‐AllprepDNA/RNAMicro(Qiagen)- Manual‐AllprepDNA/RNAMini(Qiagen)

miRNA(TotalRNAincludingmiRNA):

- Manual‐Trizol- Manual‐miRNeasyMini(Qiagen)

NB:TheQCPFhasrecentlyacquiredaQIAcube,designedtoperformfullyautomatedextractionandpurificationofnucleicacids.Theuseofourstandardizedmethods,basedonQiagenreagents,ontheQIAcubeisunderprogress.

DNA/RNAExtraction&Qualification

Date:2009‐04‐07Version:1.0

C. Qualificationofnucleicacids

Purity and integrity of nucleic acids are critical elements for the overall success of microarraysanalysis.TheRNAmustbethemostintact,withoutDNAandproteinscontamination.TheDNAmusthaveamajorbandfrom15to30kb,withoutRNAandproteinscontamination.Theassessmentofnucleicacidspurityandintegrityisperformedusingthefollowingmethods:

- MeasureoftheabsorbanceusingtheNanoDropND‐1000spectrophotometer(accesstothepurityandquantityofRNAandDNA)

- Evaluation of the electrophoretic profile using the microfluidics‐based platform AgilentBioanalyzer2100 andagarosegelelectrophoresis (access to the integrityofRNAandDNArespectively)

For eachmethod, theQCPF has definedqualification criteria, basedon literature and experience(over8000RNAand4000DNAevaluatedbetween2000and2008).

1. Puritycriteria

Ratiosevaluated:260/280and260/230PurityindicationforRNA:

Ratio Value Purityindication2 PureRNA

260/280<1,8 Presenceofproteins,phenolorothercontaminants

1,8‐2,2 PureRNA260/230

<1,8 Co‐purifiedcontaminants(organic,salts,solvents)PurityindicationforDNA:

Ratio Value Purityindication1,8 PureDNA<1,8 Presenceofproteins,phenolorothercontaminants260/280>1,8 RNAcontamination

1,8‐2,2 PureDNA260/230

<1,8 Co‐purifiedcontaminants(organic,salts,solvents)Acceptablevalues:

‐ 260/280:equaltoorgreaterthan1.8‐ 260/230:equaltoorgreaterthan1.6

2. IntegritycriteriaforRNA(AgilentBioanalyzer2100)

Threecriteriaareconsidered:

‐ 28s/18sratio‐ RIN‐ Profile

ThecombinationofthesethreecriteriadeterminesthequalificationoftheRNA.

DNA/RNAExtraction&Qualification

Date:2009‐04‐07Version:1.0

28s/18sratio:

28s/18s Level>=1,5 Good

1,3<r<1,5 Medium<1,3orNA Bad

RIN:

RIN Level>=7 Good

6<RIN<7 Medium<6orNA Bad

Profile:

Profilequalification Description LevelGoodquality Intactprofilewithaflatbaseline(RIN>=7(optimumforµarray)) GoodLowquality ProfilewithawavybaselinePartiallydegraded Smalldegradation(elevationofthewavybaselineinfastregion)Notusualprofile*

Medium

Degraded StronglyorcompletelydegradedDNAcontamination PresenceofgDNANodetection

Bad

*Theleveldependsontheprofilecomment.

Profilecomment DescriptionUndenatured Presenceofapeakafterthe28speak(dimerization)Shiftedpeaks Shiftinthesamplemigration18s/28speaksinversion Small28speak Smallanddiffuse28speak Small28speakwithalargeareaDouble28speak 28shasadoublepeakSpikes* ElectricalartefactProfilecomment:additionalinformationfordescribingtheprofile.*Spikescanbeacriticalanomalyinfastregion(noRINcomputation).Inpostregion,noncriticalanomalie(RINnumber).QualificationoftheRNA:Qualification DescriptionOK min.2/3criteriaareGoodNo min.2/3criteriaareBadLimit min.2/3criteriaareMedium+combination

ofthe3criterialevels

DNA/RNAExtraction&Qualification

Date:2009‐04‐07Version:1.0

NB:Forthesamplesqualifiedas"Limit",dependingonthecriteriacombination,thesamplescanbenotifiedasLimit(+)orLimit(‐)fortheconsortiumselection.Figure1:ElectrophoregramsofTotalRNAsamplesextractedusingManual‐RNeasyMini(Qiagen)forAetBandManual‐TrizolforC.

(A) 28s/18s=1.7;RIN=8.6;Profile=GoodQuality;Qualification=OK(B) 28s/18s=1.4;RIN=6.8;Profile=LowQuality;Qualification=Limit(C) 28s/18s=NA;RIN=2.7;Profile=Degraded;Qualification=No

(A)

(B)

DNA/RNAExtraction&Qualification

Date:2009‐04‐07Version:1.0

(C)

3. IntegritycriteriaformiRNA(AgilentBioanalyzer2100)

Ongoing...

4. IntegritycriteriaforDNA(AgaroseGelElectrophoresis)

Threecriteriaareconsidered:

‐ DNAprofile‐ Degradationlevel‐ RNAcontamination

ThecombinationofthesethreecriteriadeterminesthequalificationoftheDNA.QualificationoftheDNA:

DNAProfile DegradationLevel QualificationClearband Clearband Weaksmear

OK

Weakband Weakband Weaksmear

Limit

Weakband SmearNovisibleband Novisibleband SmearNovisibleband CompletedegradationShearedDNA

No

NB:Thebandevaluatedisthemajorbandfrom15to30kb.IncaseonRNAcontamination:Limit.

DNA/RNAExtraction&Qualification

Date:2009‐04‐07Version:1.0

Figure2:AgarosegelanalysisofgDNAsamples(E‐Gel1.2%SYBRSafe)L:ladderLambdaHindIII;1:No;2:OK;3:OK;4:OK;5:Limit

L 1 2 3 4 5