miRNeasy Serum/Plasma Kit (50)

From QIAGEN, this kit is dedicated to the purification of cell-free total RNA, including miRNA, from animal and human plasma and serum. It is designed for the recovery of primarily miRNA and other small RNA species, which in these biofluids typically consist of molecules shorter than 100 nucleotides, and processes sample volumes of up to 200 µl. Third-party product descriptions note that the workflow incorporates carrier RNA to boost recovery from low-volume specimens and that it uses inhibitor-reducing chemistry.

The chemistry combines phenol/guanidine-based lysis with silica-membrane purification. Samples are lysed in the included QIAzol Lysis Reagent, a monophasic solution of phenol and guanidine thiocyanate that facilitates lysis of tissues, inhibits RNases, and removes most cellular DNA and protein by organic extraction. After chloroform addition and centrifugation, RNA partitions to the upper aqueous phase; ethanol is then added to provide appropriate binding conditions for RNA molecules of approximately 18 nucleotides upwards. The sample is applied to an RNeasy MinElute spin column, where total RNA binds and contaminants are washed away, and the RNA is eluted in a small volume of RNase-free water. Because serum and plasma contain predominantly small RNAs, separate purification of large and small RNA fractions is not required. The same lyse, bind, wash, and elute workflow can be automated on the QIAcube.

Purified total RNA including miRNA is suitable for sensitive downstream applications, including biomarker discovery, Northern blot analysis, quantitative real-time RT-PCR with the miScript PCR System, microarray analysis, liquid biopsy miRNA profiling, and clinical research. Yields from 50–200 µl of human plasma and serum are not quantifiable by photometric methods such as NanoDrop and are usually below the limit of OD measurement; more sensitive fluorimetric or colorimetric assays such as RiboGreen are recommended. The miRNeasy Serum/Plasma Spike-In Control — a lyophilized C. elegans miR-39 miRNA mimic supplied at 10 pmol per tube and ordered separately — combined with the corresponding Ce_miR-39_1 miScript Primer Assay provides a convenient way to monitor miRNA purification and amplification. The MinElute spin columns included in the kit should be stored at 2–8 °C; short-term storage of up to four weeks at 15–25 °C does not affect performance.