Optimization of purification of human cell-free mRNA from plasma.
Cell-free RNA has a potential for diagnosis and prognosis of many diseases. Our aim was to optimize a commercially available QIAamp UltraSens Virus Kit (Qiagen, Hilden, Germany) for isolation of mRNA from human plasma. The amount of carrier RNA to bind plasma mRNA, the centrifugal force to pellet the mRNA-carrier RNA complex, the incubation time for proteolysis, and the centrifugal force to bind mRNA to the silica gel membrane were modified in order to maximize the yield of isolated mRNA. The isolated cell-free mRNA was detected for cycA using TaqMan real-time quantitative RT-PCR. The lowest threshold cycle (Ct) was obtained with the use of 4.0 microL of carrier RNA. Pelleting with a centrifugal force of 1500 xg yielded the lowest Ct, but caused difficulties in resuspending the pellet. Therefore, we suggest using a lower centrifugal force of 1300 xg. The duration of proteolysis had no effect on Ct. To bind mRNA to the silica gel membrane, a centrifugal force of 5000 xg is recommended. Our results show that the UltraSens Virus Kit is an appropriate choice for isolating mRNA from human plasma, with imprecision expressed as coefficient of variation below 2%.
Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia. tadeja.dovc-drnovsek@ztm.si
Tadeja
T
Dovc-Drnovsek
Barbara
B
Emersic
Primoz
P
Rozman
Darko
D
Cerne
Jana
J
Lukac-Bajalo
D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification*
D012333:RNA, Messenger