Principle / Procedure

NucleoSpin® 8 / 96 RNA Blood combines the stringency of guanidine-isothiocyanate lysis with effective digestion of genomic DNA by rDNase treatment and the speed of silica-membrane technology. NucleoSpin® 8 / 96 RNA Blood means consistent RNA quality, reproducible yields of RNA, and high-throughput procedures free of cross-contamination. Therefore, NucleoSpin® 8 / 96 RNA Blood kits are powerful products for, for example, microarray analysis in respect to gene expression profiling, drug discovery, and molecular diagnostics. The kits allow purification of total RNA from up 400 µL whole blood. The kit content is primarily focused on vacuum processing. For centrifugation, suitable consumables for waste collection have to be ordered separately (see consumables supplied by user). During the NucleoSpin® 8 / 96 RNA Blood method, blood cells are directly lysed by incubation in a solution containing large amounts of chaotropic ions. This lysis buffer immediately inactivates RNases and creates appropriate binding conditions which favor adsorption of RNA to the silica membrane of the NucleoSpin® 8 RNA Blood Binding Strip / 96 RNA Blood Binding Plate.
Contaminating DNA, which is also bound to the silica membrane, is removed by an on-column DNA digestion. Due to the highly specific activity of the rDNase, a treatment of only 15 minutes at room temperature is sufficient for an efficient reduction of contaminating DNA (see application data). Salts, metabolites, and macromolecular cellular components are removed by simple washing steps with three different buffers. Pure RNA is finally eluted under low ionic strength conditions with RNase-free water.

Required hardware

Vacuum processing
The NucleoSpin® 96 RNA Blood kit can be used with the NucleoVac 96 Vacuum Manifold (see ordering information). When using NucleoSpin® 96 RNA Blood with less than 96 samples, Self-adhering PE Foil (see ordering information) should be used in order to close and protect non-used wells of the NucleoSpin® 96 RNA Blood Binding Plate and thus guarantee a proper vacuum.
The NucleoSpin® 8 RNA Blood kit can be used manually with the NucleoVac 96 Vacuum Manifold (see ordering information) by using the Starter Set A. This Starter Set contains two Column Holders A and 12 NucleoSpin® Dummy Strips . Starter Set A is also required for automation on laboratory platforms with standard 96-well plate vacuum chambers.

Centrifugation
For centrifugation a microtiterplate centrifuge is required. This centrifuge must be able to accommodate either the NucleoSpin® RNA Blood Binding Plate stacked on a round or square-well block or the NucleoSpin® RNA Blood Binding Strips stacked on a square-well block (bucket height: 85 mm). A suitable centrifuge must be capable to reach accelerations of 5,600–6,000 x g. In addition, for the usage of NucleoSpin® 8 RNA Blood Binding Strips Starter Set C (see ordering information) is required. Starter Set C contains two Column Holders C, two MN Square-well Blocks, and two Racks of Tube Strips.

Consumables to be supplied by user
NucleoSpin® 8 / 96 RNA Blood – centrifuge processing: For waste collection, suitable consumables (e.g., MN Square-well Blocks) are necessary and not included in the kits. For the most convenient handling, without reusing the MN Square-well Blocks, we recommend using six MN Square-well Blocks if two Column Holders C (NucleoSpin® 8 RNA Blood) or if two 96-well plates (NucleoSpin® 96 RNA Blood) are processed in parallel. Alternatively, it is possible to empty the MN Square-well Blocks after every centrifugation step, reducing the total amount of MN Square-well Blocks needed.

NucleoSpin® 8 / 96 RNA Blood procedure

Features

* RNA yield strongly depends on the leukocyte number in each individual blood sample.

Application data
    
High yield and quality – very convenient handling – high flexibility in sample volume and sample type
NucleoSpin® RNA Blood kits are based on a simple and convenient direct blood lysis. This procedure allows a very effective blood cell lysis at room temperature without an upstream selective erythrocyte lysis at 4 °C. Compared with competitor kits, NucleoSpin® RNA Blood kits show higher yields from smaller sample volume. In addition it is possible to obtain a linear increase of yield in regard to sample volume. Both fresh and frozen blood samples can be used to purify RNA with comparable yield and quality.

Consistent RNA yield and quality from fresh or frozen blood samples
NucleoSpin® 96 RNA Blood has been used to isolate RNA in triplicate from 400 μL fresh and frozen blood (EDTA) samples under vacuum. Samples originated from six individual blood donors. Frozen sample aliquots have been stored at –20 °C. RNA yield and quality were determined by spectrophotometric measurement of absorption at 260 nm and 280 nm. The diagrams show the average yield (A) and A260/A280 ratio (B) calculated for every triplicate.

High quality RNA
RNA has been isolated out of 400 µL aliquots of fresh whole blood from three individual blood samples using NucleoSpin® 8 RNA Blood. Electropherograms were obtained from the Agilent Bioanalyzer 2100 using RNA Nano 6000 reagents. The RNA integrity number (RIN) ranged from 8.0 to 8.30.
    


Effective DNA Removal
Total RNA was isolated manually under vacuum from 400 µL whole blood samples (EDTA) according to the standard protocol for NucleoSpin® 96 RNA Blood with and without an on-column rDNase treatment. A PCR for human elongation factor EF-1a has been performed to determine the amount of residual genomic DNA in the eluted RNA. The figure shows the corresponding amplifilication curves of the 0.01–100 ng DNA standards (dark blue lines), the NTC (negative control; green), duplicates of blood sample 1 (1-1, 1-2) with DNase treatment (+ DNase; red lines) and without DNase treatment (-DNase; light blue lines).
In total, blood samples from six individual donors have been analyzed by PCR (data not shown). Samples treated with rDNase (+DNase) show an average Cp of 31.8. Samples purified without rDNase (-rDNase) treatment show an avreage Cp of 25.9. Omitting the on column DNA digestion results in an approximately 64 times higher amount of residual gDNA.

Analysis of DNA with LightCycler® PCR, human elongation factor EF-1a specific primer, 469 nt amplification target.