| Abstract | Izolacija DNA je ključni korak koji prethodi brojnim analizama u molekularnoj dijagnostici. Izlacija DNA iz leukocita isoljavanjem (po Milleru ili Qiagen test paketom) temelji se na odvajanju staničnih proteina povećanjem koncentracije soli i centrifugiranjem, nakon čega se DNA taloži apsolutnim etanolom. Cilj istraživanja je ispitati utjecaj broja leukocita na koncentraciju i čistoću izolirane DNA.
Broj leukocita određen je neposredno nakon uzorkovanja na hematološkom brojaču. Uzorci pune krvi pomiješani s antikoagulansom EDTA su pohranjeni na -20° C do izolacije DNA. DNA je izolirana metodom po Milleru (N=227; 92 zdrava ispitanika i 135 ispitanika s kroničnom opstrukcijskom plućnom bolesti) i primjenom komercijalnog kita tvrtke Qiagen (N=173; 98 zdravih ispitanika i 75 ispitanika na hemodijalizi). Apsorbancije na 280 i 260 nm izmjerene su na DeNovix DS-11 spektrofotometru visoke osjetljivosti. Statistička analiza provedena je korištenjem MedCalc statističkog programa.
Millerovom metodom izolacije u odnosu na Qiagen test paket dobivene su veće koncentracije DNA [138,6 (123,1-151,7) vs 57,0 (48,0-76,5) ng/μL; P<0,001] i čistoće [1,7 (1,7-1,7) vs 1,3 (1,3-1,5); P<0,001]. Broj leukocita se značajno razlikovao u uzorcima korištenim za dvije različite metode izolacije. Pronađena je slaba korelacija između broja leukocita i koncentracije DNA (rs=0,27; P<0,001 i rs=0,43; P<0,001) i između broja leukocita i čistoće DNA (rs=0,38; P<0,001 i rs=0,27; P<0,001).
Ovisno o korištenoj metodi broj leukocita je utjecao na koncentraciju i čistoću izolirane DNA, međutim uočena korelacija iako značajna je vrlo slaba. Svakako treba uzeti u obzir da su uzorci prije izolacije zamrznuti, što je moglo utjecati na dobivene rezultate. |
| Abstract (english) | Isolation of DNA is a key step that precede various analysis in molecular diagnostics. Extracting DNA from leukocytes by salting out method (by Miller or with Qiagen purification kit) is based on increasing salt concentration which separates proteins and cellular debris from the DNA fraction. After centrifugation of the mixture, DNA molecules are precipitated by absolute ethanol. Purpose of this research is to examine the impact of number of leukocytes on concentration and purity of isolated DNA.
Number of leukocytes was determined on automated hematology analyser immediately after sampling of blood. Whole blood samples taken on anticoagulant EDTA were stored on -20 °C until DNA extration. DNA was isolated by Miller method (N=227; 92 healthy subjects and 135 subjects with chronic obstructive pulmonary disease) and Qiagen commercial kit (N=173; 98 healthy subjects and 75 patients undertaking hemodialysis). Apsorbances at 260 and 280 were measured on DeNovix DS-11 highly sensitive spectrophotometer. Statistic analysis was done by MedCalc statistic program.
Higher DNA concentrations were isolated by Miller method compared to the Qiagen comercial kit method [138.6 (123.1-151.7) vs 57.0 (48.0-76.5) ng/μL; P<0.001 and purity [1.7 (1.7-1.7) vs 1.3 (1.3-1.5); P<0.001]. Number of leukocytes in samples used for two methods of isolation was significantly different. We observe weak correlation between number of leukocytes and DNA concentration (rs=0.27; P<0.001 and rs=0.43; P<0.001) and between number of leukocytes and DNA purity (rs=0.38; P<0.001 and rs=0.27; P<0.001).
Depending on method, number of leukocytes showed significant but weak impact on extracted DNA concentration and purity. However, it is very important to know that before isolation all samples were frozen, which could have an effect on the obtained results. |
