Naslov (srp)

Uticaj formalina i parafinskog bloka na fragmentaciju molekula DNK u humanim tkivima izuzetih prilikom izvođenja sudskomedicinskih obdukcija

Autor

Vitošević, Katarina, 1983-, 21977447

Doprinosi

Todorović, Danijela, 1969-, 13821799
Milošević-Đorđević, Olivera, 1963-, 13622375
Alempijević, Đorđe, 1967-, 12732775
Sazdanović, Maja, 1974-, 13578599

Opis (srp)

Cilj ove studije je bio da se ispita efekat formalina i parafinskog bloka na kvalitet DNK izolovane iz različitih zdravih tkiva izuzetih tokom sudskomedicinskih obdukcija. Korišćena su zdrava tkiva srca, jetre, mozga, pluća i bubrega koja su fiksirana u 10% puferizovanom ili 4% nepuferizovanom formalinu od 6h do 2 meseca, kao i parafinski blokovi zdravih tkiva srca, jetre i mozga arhivirani od 1988-2017. godine. Morfološke odlike tkiva su proučavane nakon H&E bojenja preparata. DNK je izolovana pomoću fenol-hloroform-izoamil alkohola (PCI) i komercijanog kita. Čistoća i prinos DNK su određeni spektrofotometrijski. Integritet izolovane DNK je određen PCR amplifikacijom GPD1 (150bp), ACTB (262bp) i RPL4 (407bp) gena. Na histomorfološke karakteristike tkiva ne utiču ni fiksiranje tkiva u formalinu ni kalupljenje u parafinski blok. Ekstrakcija DNK iz uzoraka tkiva PCI metodom omogućuje dobijanje DNK boljeg kvaliteta od DNK dobijene korišćenjem kita. Bolji kvalitet DNK je u uzorcima tkiva fiksiranih u 10% puferizovanom formalinu. Ne postoji razlika u čistoći i integritetu DNK između različitih fiksiranih tkiva, već samo u prinosu: najveći prinos DNK je dobijen u uzorcima iz tkiva jetre, a najmanji iz srca i mozga. Dužina fiksiranja utiče na prinos i integritet DNK: do 72h fiksiranja je moguće amplifikovati fragmente dužine do 407bp, a do 14 dana fragmente do 262bp. Najveći prinos DNK je dobijen iz ukalupljenog tkiva jetre, a iz tkiva srca i mozga je izlovana DNK očuvanijeg integriteta. Dužina arhiviranja parafinskih blokova tkiva utiče na kvalitet DNK: RPL4 je amplifikovan do 6 godina, ACTB do 26 godina i GPD1 do 30 godina arhiviranja parafinskih blokova tkiva. DNK izolovana PCI metodom iz tkiva srca i mozga koja su fiksirana u 10% puferizovanom formalinu ima najbolji kvalitet, pa su ova tkiva pogodana za molekularno biološke analize i nakon 30 godina arhiviranja.

Opis (eng)

The aim of this study was to examine the effect of formalin and paraffin block on the quality of DNA isolated from various healthy tissues excluded during medico-legal autopsies.Healthy heart, liver, brain, lung and kidney tissues fixed in 10% buffered or 4% unbuffered formalin from 6h to 2 months were used, as well as paraffin blocks of healthy heart, liver and brain tissues archived from 1988-2017. Morphology of tissues was studied using H&E staining. DNA was isolated using phenol-chloroform-isoamyl alcohol (PCI) and commercial kit. The purity and yield of DNA were determined spectrophotometrically. The integrity of the isolated DNA was determined by PCR amplification of GPD1 (150bp), ACTB (262bp) and RPL4 (407bp) genes. The histomorphological characteristics of the tissue are not affected by tissue fixation in formalin or embedding into a paraffin block. Extraction of DNA from tissue samples by PCI method enables obtaining better quality of DNA than DNA obtained using commercial kit. Better DNA quality is obtained from samples isolated from tissues fixed in 10% buffered formalin. There is no difference in the purity and integrity of DNA between different tissues, but only in yield: the highest DNA yield was obtained in samples from fixed liver tissues, and the lowest from the heart and brain. The duration of fixation аffects on yield and integrity of DNA: fragments up to 407bp in length can be amplified up to 72h of fixation, and fragments up to 262bp in length up to 14 days of fixation. The highest yield of DNA was obtained from embedded liver tissue and DNA of more preserved integrity was extracted from heart and brain tissue. The duration of archiving of paraffin tissue blocks аffects the quality of DNA: RPL4 is amplified up to 6 years, ACTB up to 26 years and GPD1 up to 30 years of archiving paraffin tissue blocks. DNA isolated by PCI method from heart and brain tissues fixed in 10% buffered formalin has the best quality, so these tissues are suitable for molecular biological analysis even after 30 years of archiving.

Jezik

srpski

Datum

2021

Licenca

Ovo delo je licencirano pod uslovima licence
Creative Commons CC BY-NC-ND 2.0 AT - Creative Commons Autorstvo - Nekomercijalno - Bez prerada 2.0 Austria License.

CC BY-NC-ND 2.0 AT

http://creativecommons.org/licenses/by-nc-nd/2.0/at/