NucleoSpin RNA Blood
NucleoSpin RNA Blood kits provide high yields of high-quality total RNA using a simple, direct blood cell lysis procedure which is very effective at room temperature, without the need for an upstream selective erythrocyte lysis at 4°C (Figure 1). These kits are available in convenient miniprep, midiprep, 8-well strip, and 96-well plate formats that offer high flexibility with regard to sample volume and sample type.
Figure 1. The NucleoSpin RNA Blood procedure.
NucleoSpin RNA Blood and NucleoSpin RNA Blood Midi kits were shown to provide higher yields of high-quality RNA from smaller sample volumes than competitor kits (Figure 2).
Figure 2. Higher yields from smaller sample volumes than 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).
The yield of NucleoSpin RNA Blood kits was shown to increase linearly with increased sample volume (Figure 3), indicating the efficiency and reliability of the kit protocol.
Figure 3. Proportional increase in yield with increasing sample volume. NucleoSpin RNA Blood Midi was used to isolate RNA from 400-µl, 700-µl, and 1,300-μl blood (EDTA) aliquots obtained from three different donors (Blood A–C). Yields increased in proportion to the sample volume used, indicating the high efficiency and reliability of this procedure.
Fresh and frozen blood samples were used to purify RNA of comparably high yield and quality (Figure 4).
Figure 4. Consistent RNA yield and quality from fresh or frozen blood samples. NucleoSpin RNA Blood was 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 were 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 (Panel A) and A260/A280 ratio (Panel B) calculated for each triplicate sample.
Analysis of RNA purified from aliquots of fresh whole blood using NucleoSpin 8 RNA Blood indicated that the kit consistently yielded high-quality samples (Figure 5).
Figure 5. High-quality RNA. RNA was isolated from 400-μl aliquots of fresh whole blood from three individual blood samples using NucleoSpin 8 RNA Blood. Electropherograms were generated using an Agilent Bioanalyzer 2100 with RNA Nano 6000 reagents. The RNA integrity number (RIN) ranged from 8.0 to 8.3.
PCR analysis of RNA isolated manually under vacuum using NucleoSpin 96 RNA Blood indicated that inclusion of the on-column DNase treatment in the kit protocol resulted in a ~64X reduction of gDNA in the eluted RNA (Figure 6).
Figure 6. Effective DNA removal. Total RNA was isolated manually under vacuum from 400-μl samples of whole blood according to the standard protocol for NucleoSpin 96 RNA Blood, either with or without the inclusion of the on-column DNase treatment (+DNase or -DNase, respectively). The purified RNA samples were then analyzed by qPCR, along with a negative control sample (NTC) and a dilution series of DNA standards (0.01–100 ng) using the human elongation factor EF-1a as an amplification target. In total, blood samples from six individual donors were processed in this manner (data not shown). RNA samples treated with DNase yielded a Cp value of 31.8 on average, while untreated samples yielded an average Cp value of 25.9, suggesting that inclusion of the on-column DNase treatment in the kit protocol resulted in a ~64X reduction of gDNA in the eluted RNA.
Direct lysis using a NucleoSpin RNA Blood kit was shown to provide higher yields of RNA than the selective erythrocyte lysis protocol of a competitor kit (Figure 7).
Figure 7. Direct lysis results in higher yields than selective erythrocyte lysis. RNA was isolated from 400 μl of 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 were analyzed in duplicates. For both samples, Ct values were lower for NucleoSpin RNA Blood, indicating a higher RNA yield. The isolated RNA was analyzed with LightCycler RT-PCR using a β-actin-specific primer and a 73-nt amplification target.
| NucleoSpin RNA Blood | NucleoSpin RNA Blood Midi | NucleoSpin 8/96 RNA Blood | |
| Technology | Silica membrane | Silica membrane | Silica membrane |
| Format | Mini spin columns | Midi spin columns | 8-well strips/ 96-well plates |
| Starting material | <400 µl of whole blood (fresh or frozen) |
400–1,300 µl of whole blood (fresh or frozen) | <400 µl of whole blood (fresh or frozen) |
| Fragment size | >200 nt | >200 nt | >200 nt |
| Typical yield | 1–8 µg (400 µl of whole blood)* |
4–26 µg (1,300 µl of whole blood)* |
1–8 µg (400 µl of whole blood)* |
| A260/A280 | 1.9–2.1 | 1.9–2.1 | 1.9–2.1 |
| Elution volume | 40–120 µl | 200–400 µl | 50–130 µl |
| Preparation time | 55 min/6 preps | 75 min/6 preps | 60 min/6 strips 100 min/plate |
| Binding capacity | 200 µg | 700 µg | 100 µg |
*RNA yield depends on the leukocyte number in each individual blood sample.
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