Molekler Testlerde Preanalitik Faz

Molekler Testlerde Preanalitik FazProf. Dr. Abdulkerim BEDROndokuz Mays niversitesiSamsun2014Lab Dngs

HastaEdinim/Acquisitionlem/ProsesSaklamaDatmBilimsel Analiz

Tbbi/CerrahiProsedrlerArtanStoklamaBiyospesmen and sreBiyospesmen BilgilerininDerleme ve AnaliziKlinik/Klinik Aratrma ktlarBilimsel BilgilerinDerleme ve Analizi3Molekler Ama Genomics Proteomics Metabolomics

Yksek kalitede Biyospesmenlere baldr1-Tehis etmek2-Tedavi belirlemek3-Risk analizi4Molekler deal

BugnYarnThree colon cancer patients: Same disease? Same therapy?

Each patient differs with respect to the molecular basis of his/her cancer

5Molekler Evren

Molekler Doma

Molekler Ak

Preanalitik faktrler I: Sv BiyospesmenAntikoaglanlarSitrat-DNA, RNAEDTA-DNAHeparin-Sitolojik testlerStabilizr/nhibitrlerProtein: Proteaz inhibitrleriRNA: Beta-merkaptoetanol (stabilizr)RNaz inhibitrleriDNA: Stabildir-rn. Guthrie testiSaklama scaklPMSF, Leupeptin, pepstatin, aprotinin

2-Mercaptoethanol is used in some RNA isolation procedures to eliminate ribonuclease released during cell lysis. Numerous disulfide bonds make ribonucleases very stable enzymes, so 2-mercaptoethanol is used to reduce these disulfide bonds and irreversibly denature the proteins. This prevents them from digesting the RNA during its extraction procedure9Bekleme sresiHcre saym 24 saatte derSteriliteBakteriyal kontaminasyonFungal kontaminasyonEndojen bozucu etkenlerEnzimler: Proteazlar, DNazlar, RNazlarHcre lmNumune tpleri/kaplarDNaz-freeRNaz-freeSterilAntikoagulanlarAditifRenkTestlerYokKrmzBiyokimya, Seroloji, immnoloji, serumSodium heparin (freeze dried)YeilImmnoloji, viroloji testleri Sodium heparinKahverengiSitogenetik testler, molekler testler Tripotassium EDTA (7.5-15% solution)MorVirology, molecular biologyAcid citrate dextrose (ACD) solutionSarMolekler biyoloji11Preanalitik faktrler II:Solid BiyospesmenKanser hcrelerinde genomik deiiklikler dk frekanstadrKantite sorunuBiyopsi materyaliKalite sorunuBiyopsi materyalleri formalinle fiksedir. DNA izolasyonu zel protokole tabidirPrite sorunu (Tmr heterogenitesi)Normal hcreler ile karktrKanserin kendisinde heterogenite (farkl klonlar)RNAlater RNA Stabilization Reagent immediately stabilizes RNA in tissue samples to preserve the gene expression profile, and is supplied in 50 ml or 250 ml bottles. For stabilization of DNA, RNA, and protein in tissue samples, Allprotect Tissue Reagent can be used. 12FFPE (formalin-fixed, paraffin-embedded) rneklerFiksasyon sresiParafine gmme scaklDoku takip ilemiParafin bloklar saklama artlarDNA intak deildirKovalen addkler oluurPCR 300 bp fragmandifferences (characteristics of formalin fixation; duration of fixation, type of tissue processing, temperature of paraffin embedding, storage conditions of paraffin blocks)

For many retrospective studies, formalin-fixed and paraffin-embedded ... fail to allow for effective amplification of DNA fragments beyond 300 bp

However, nucleic acids isolated from FFPE tissues are severely degraded and contain mainly small fragments, generally less than 300 bp. These fragments represent a poor substrate for molecular biological methods, e.g. PCR [1,2]. Furthermore, formalin-fixation leads to the formation of DNA-protein crosslinks, which are not completely removed by common lysis protocols [3]. Crosslinks increase the sensitivity of DNA to mechanical stress and decrease the accessibility for enzymes. In addition, formalin is oxidized to formic acid which causes DNA depurination and DNA strand breaks.

Fresh or fresh frozen tissue samples are the best material for isolation DNA of high quality and quantity. However, storage of frozen tissue samples is expensive and time-consuming. For many retrospective studies, formalin-fixed and paraffin-embedded (FFPE) material is, therefore, the only available tissue for DNA analysis. Isolation of sufficient amounts of intact DNA from FFPE tissue samples is challenging. One major problem is DNA-protein cross-linking caused by formalin fixation that must be broken up during the extraction process.3,8 Furthermore, the low pH in unbuffered fixatives leads to degradation of most DNA molecules into fragments of 200 bp or less.3 A number of approaches have been published to deal with these problems that hinder the use of FFPE for genomic analysis.1,2,57,9 These protocols use expensive DNA extraction kits,2,5,7,9 have low DNA yield,2,6 have suboptimal purity of DNA,1 or fail to allow for effective amplification of DNA fragments beyond 300 bp.1,2,5,6 13Taze doku rnekleriPatolog gzetiminde allmaldrnce ine biyopsi materyalleriHcre says yetersiz olabilir

Saklama koullar- DNANumunelerKan, Kemik ilii, Vcut svlar1 year, -20C or -70C Doku2 hafta, -20C >2 yl, -70CIzole DNA