Principle / Procedure

The NucleoSpin® RNA Blood procedure is a simple and convenient method to isolate RNA from fresh or frozen whole blood stabilized, for example, with EDTA, citrate, or heparin.
The simple and efficient direct total blood lysis allows RNA isolation from whole blood without the common but cumbersome selective erythrocyte lysis at 4 °C. Thus the complete procedure is performed at room temperature. Blood cells are lysed in Lysis Buffer DL. The RNA is bound to the NucleoSpin® RNA Blood Binding Column. DNA is efficiently removed by an on-column rDNase digestion. Contaminants and inhibitors are removed with two different wash buffers. Finally RNA is eluted with RNase-free water.

Features

Technology: Silica-membrane technology
Format: Mini spin columns
Sample material: Up to 400 µL whole blood (fresh or frozen)
Fragment size: > 200 nt
Typical yield: 1.2–8 µg (400 µL whole blood)*
Typical purity (A260/A280): 1.9–2.1
Elution volume: 40–120 µL
Preparation time: 55 min/6 preps
Binding capacity: 200 µg
    
* 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.

	Higher yield from smaller sample volume – comparison to competitor kits RNA was isolated from the indicated blood volumes following each manufacturer's protocol. The quality of isolated RNA is comparable. Average RIN numbers (data not shown) were: 7.5 (Q); 7.7 (NucleoSpin® RNA Blood); 8.8 (NucleoSpin® RNA Blood Midi); 8.9 (Q, PAXgene®).
	Direct lysis results in higher yields compared to selective erythrocyte lysis RNA was isolated from 400 μL blood (EDTA) from two different donors (Blood A, B) with the NucleoSpin® RNA Blood kit and a kit from Competitor Q (based on selective erythrocyte lysis). Samples have been analyzed in duplicates. For both samples, Cp values are lower for NucleoSpin® RNA Blood indicating a higher RNA yield. Analysis of RNA with LightCycler® RT-PCR, β-actin specific primer, 73 nt amplification target.
	Double yield for 400 µL blood RNA has been isolated out of  200 µL and 400 µL aliquots from 9 individual blood samples using NucleoSpin® RNA Blood. Using the 400 µL aliquot, the yield was doubled in average.

(A) Bioanalyzer analysis	(B) Quantitative RT-PCR analysis

Isolation and amplification of high quality RNA from blood samples stored under different conditions
Identical aliquots of 12 individual blood samples have been stored under different conditions. RNA isolation has been performed with NucleoSpin® RNA Blood.
Condition 1: no storage; RNA has been isolated directly from aliquots of 200 µL fresh whole blood.
Condition 2: aliquots of 200 µL whole blood were stored for 5 days at -20 °C.
Condition 3: 200 µL Lysis Buffer DL was added to 200 µL whole blood and mixed. The resulting lysates were stored for 5 days at 4 °C.
Condition 4: lysates (as described in condition 3) were stored for 5 days at -20 °C.
The average RNA quality (RNA integrity number = RIN) is hardly affected by different storage conditions (A). The isolated RNA shows a similar high performance in qRT-PCR (B) regardless of the different storage conditions. Red bars indicate the average RIN (A) or the average Cp value (B) respectively. Error bars represent standard deviations.


Analysis of RNA with LightCycler® RT-PCR, β-actin specific primer, 73 nt amplification target.