DNA/RNA Extraction & Qualification Date : 2009‐04‐07 Version : 1.0 The Quality Control Platform (QC PF) of Saint‐Louis, funded by the «Ligue nationale contre le cancer», is dedicated to the quality control of biological resources in the framework of the CIT program (Platform directed by Dr Mira AYADI ‐ [email protected]). Process: A. Collection of the consortium samples (tissue, cells, lysate, DNA, RNA, miRNA) B. Extraction of nucleic acids (if required) C. Qualification of nucleic acids for microarrays and/or real‐time PCR applications A. Collection of the consortium samples The identification of the samples is controlled or performed according this format <project alias> _ <projetc investigator alias> _ <species alias > _ <sample number> . All information about the samples are collected in a database (database software: FileMaker Pro) to ensure the traceability. Storage conditions: ‐ Tissue, cells, lysate, RNA and miRNA are stored at ‐80°C ‐ DNA are stored at ‐20°C B. Qualification of nucleic acids Nucleic acids are extracted based on standardized methods: DNA: - Manual‐Phenol/Chloroform - Manual‐QIAamp DNA Mini (Qiagen) RNA: - Manual‐Trizol - Manual‐RNeasy Micro (Qiagen) - Manual‐RNeasy Mini (Qiagen) DNA/RNA simultaneously: - Manual‐Allprep DNA/RNA Micro (Qiagen) - Manual‐Allprep DNA/RNA Mini (Qiagen) miRNA (Total RNA including miRNA): - Manual‐Trizol - Manual‐miRNeasy Mini (Qiagen) NB : The QC PF has recently acquired a QIAcube, designed to perform fully automated extraction and purification of nucleic acids. The use of our standardized methods, based on Qiagen reagents, on the QIAcube is under progress.
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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:
Purity and integrity of nucleic acids are critical elements for the overall success of microarraysanalysis.TheRNAmustbethemostintact,withoutDNAandproteinscontamination.TheDNAmusthaveamajorbandfrom15to30kb,withoutRNAandproteinscontamination.Theassessmentofnucleicacidspurityandintegrityisperformedusingthefollowingmethods:
- 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).