dna/rna extraction & qualification - le...
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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‐[email protected]).
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
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