Optimization of Purification of Human Cell‐Free mRNA from Plasma

Abstract

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%.

Circulating nucleic acids in plasma and serum: diagnosis and prognosis in cancer

“…Either the blood can be held for various periods of time [19, 20] or the serum/plasma are removed immediately from whole blood and stored [21] or the nucleic acids are removed and stored for further study. Cellular debris is removed by centrifugation either once or more so as to remove any cellular DNA/RNA [22, 23]. Filtering has been used, but nucleic acids can stick to the filter and so be lost from the material to be analyzed.…”

Biology of Circulating Nucleic Acids and Possible Roles in Diagnosis and Treatment in Diabetes and Cancer

“…Sometimes the blood is held for various periods of time [44] or the serum/plasma are removed and stored [141] or the nucleic acids are removed and stored for further study. Blood is centrifuged once or more to remove cellular debris to avoid the presence of cellular DNA/RNA [142,143]. Filtering has been used, but nucleic acids can stick to the filter and the removal of any cells will remove cell surface-bound nucleic acid fraction.…”

Frontiers in Preclinical Safety Biomarkers

“…Kits/methods are optimized for the extraction of small RNAs either in conjunction with full length total RNA or as a fraction enriched for small RNAs. Currently available kits include mirVana 2 miRNA isolation kit (Life Technologies, Austin, TX), miRNAeasy kit (Qiagen, Valencia, CA), Trizol 1 LS method (Life Technologies, Carlsbad, CA), the QIAamp 1 UltraSens 2 Virus kit (Qiagen), and Exiqon's Locked Nucleic Acid 2 microarray platform (Vedbaek, Denmark; Mraz et al 2009;Dovc-Drnovsek et al 2008). The method used in our laboratory combines Trizol LS with column-based RNA isolation.…”