Focus: Isolation and Analysis of Nucleic Acids

Can the SV RNA Isolation System be used to Purify RNA from Frozen, Unprocessed Whole Blood?

Rapid processing of blood specimens is critically important for maximizing the yield and quality of isolated nucleic acids (1). However, the appropriate processing of blood samples may be impractical or impossible in samples such as stored blood. This report demonstrates the isolation of total RNA from unprocessed frozen whole blood using Promega's SV Total RNA Isolation System (Cat.# Z3100). While isolation of RNA from unprocessed frozen blood is possible, freezing of the whole blood leads to lower RNA yields and inferior performance in RT-PCR when compared to RNA isolated from frozen samples that had been treated to remove red cells and other non-leukocyte material.

By Daniel Kephart, Ph.D., and Hemanth Shenoi, Ph.D.
Promega Corporation

Published in September, 1999

Introduction

Whole human blood is often used as a convenient and rich source of nucleic acids for analysis of disease states and genetic testing in both research and clinical diagnostic laboratories. The cellular components of blood include erythrocytes and leukocytes with leukocytes as the primary target in most cellular nucleic acid isolation strategies. Isolation of these cells requires anticoagulated blood, and removal of the highly abundant erythrocytes is required in order to maximize nucleic acid yields from the desired leukocyte population. As little as 1% of whole blood (v/v) can inhibit PCR, possibly due to the binding of heme or porphyrin to Taq DNA polymerase (2). Therefore, lysis and removal of red blood cell components during nucleic acid isolation is critical when preparing material for downstream applications involving PCR. In addition, the wholesale release of nucleases and other contaminants within the blood sample may lead to lower RNA yields and increased protein contamination.

According to guidelines published by the American Association for Clinical Chemistry (AACC), investigators seeking to obtain RNA should not freeze whole blood (3). Freezing results in the lysis of all blood cells and can result in significant decreases in the quantity and quality of isolated nucleic acids, especially RNA. A simple pretreatment of whole blood samples to remove erythrocytes and plasma prior to freezing of leukocytes can greatly increase the quality of isolated RNA relative to RNA isolated from blood stored under suboptimal conditions (1). However, the need to analyze inappropriately processed or archived blood samples may necessitate the isolation of RNA from frozen whole blood. In this case, one must consider several parameters including; 1) how to process frozen whole blood, 2) how total RNA yield may be affected by freezing of whole blood, and 3) how freezing may affect the quality of the RNA isolated as evidenced by the ablility to detect the target RNA.

Experimental Conditions

Whole human blood was collected by venipuncture into EDTA-coated Vacutainer^® tubes for RNA isolation. Total RNA was isolated within 2 hours at ambient temperature from 200µl aliquots of fresh whole blood following the protocol for red blood cell lysis and leukocyte processing provided with the SV Total RNA Isolation System (4). For RNA isolation from frozen blood, a 1ml aliquot of whole blood was frozen at –80°C for 24 hours. Freezing of the whole blood sample resulted in lysis of all cells in the sample, making removal of red blood cell contaminants or concentration of leukocytes impossible prior to RNA isolation. As such, 200µl portions of the frozen whole blood were processed by adding 175µl of SV RNA Lysis Buffer directly to the thawed sample before processing using the standard SV Total RNA Isolation System protocol.

RT-PCR was performed using Promega's Access RT-PCR System (Cat.# A1250). Reactions were performed according to the protocol given in the Access RT-PCR System Technical Bulletin #TB220. See Figure 1 for primer information and amplification conditions.

Results

RNA yield and purity were first determined by spectrophotometric analysis and averaging of five independent isolations from the same blood sample (Table 1).

Isolation of RNA from fresh blood yielded 3.6 times more RNA than was obtained from frozen samples stored overnight at –80°C. The freezing of whole blood also led to increased protein contamination in the RNA, indicated by the 33% decrease in the A₂₆₀/A₂₈₀ ratio relative to RNA isolated from fresh blood. Lysis of red blood cells in the frozen sample also led to increased heme contamination when the final RNA samples were inspected visually.

Samples were then analyzed by RT-PCR to determine if the quality of RNA isolated from frozen whole blood was compromised relative to RNA isolated from fresh blood. In this experiment, RNA isolated from fresh or frozen blood was precipitated and resuspended to 50ng/µl in Nuclease-Free Water. Decreasing amounts of the RNA isolated from fresh or frozen blood were then analyzed by RT-PCR amplification of three different targets (Figure 1).

Figure 1. RT-PCR amplification of total RNA isolated from human blood. Total RNA isolated from fresh or frozen whole human blood was analyzed by RT-PCR. Aliquots of isolated RNA were precipitated using nuclease-free glycogen as a carrier, resuspended to equal RNA concentrations in Nuclease-Free Water, and the indicated amounts of total RNA isolated from fresh whole blood (lanes 1–3) or frozen whole blood (lanes 4–6) were used as template with the Access RT-PCR System (4). Primer pairs for a segment of the beta-actin gene (Panel A), a segment of the adenomatous polyposis coli gene (Panel B), or a segment of the ERK1 gene (Panel C) were used to determine the relative quality of RNA isolated from fresh or frozen blood samples. Lane 7 of each panel is a reaction lacking reverse transcriptase to demonstrate that the signal observed was derived from RNA. Equivalent aliquots of each amplification reaction were analyzed by agarose gel electrophoresis. Cycling conditions were 1 × (45 minutes at 48°C); 1 × (2 minutes at 94°C); 40 × [(0.5 minute at 94°C), (1 minute at 60°C), (1 minute at 68°C)]; 1 × (7 minutes at 68°C); 4°C soak. Lane M, Promega's 100bp DNA Ladder (Cat.# G2101).

The analysis of equivalent amounts of total RNA indicated that the RNA isolated from frozen blood samples did not perform as well in amplification as that isolated from fresh blood. The amplification signal strength using a primer pair designed to amplify a portion of the abundant beta-actin mRNA was significantly lower but detectable at all concentrations tested. Although RT-PCR detection of RNAs encoding a portion of two less abundant RNAs including the adenomatous polyposis coli and ERK1 gene products was easily accomplished using 50ng of RNA isolated from fresh blood, detection of these targets was almost completely eliminated in RNA isolated from frozen whole blood (Figure 1).

Conclusions

We investigated the use of frozen whole human blood as a source for RNA isolation using Promega's SV Total RNA Isolation System. The freezing of whole blood leads to lysis of all cells, precluding the selective lysis and elimination of reticulocytes, as well as a step to concentrate the leukocytes present in the sample. We demonstrated that 200µl of frozen whole blood can be used successfully with the SV Total RNA Isolation System. However, the quantity and quality of the RNA isolated from frozen blood is significantly diminished relative to RNA isolated from fresh anticoagulated blood. A significant decrease in amplification performance was observed after storage of frozen blood overnight at –80°C. This effect is expected to increase with the greater length of storage encountered with some archived samples.

References

1. Kephart, D. and Shenoi, H. (1998) Molecular Diagnostics: Isolation and analysis of RNA from human blood. Promega Notes 68, 23.
   
2. Higuchi, R. (1995) Simple and rapid preparation of samples for PCR In: PCR Technology: Principles and Applications for DNA Amplification, Erlich, H.A., ed., Stockton Press, New York, 31.
   
3. Farkas, D.H., Drevon, A. M. and DiCarlo, R.G. (1997) Molecular Pathology: Basic Methodology and Clinical Application. AACC Self-Study Course Manual. Klosinski, D. ed., AACC, Washington DC.
   
4. SV Total RNA Isolation System Technical Manual #TM048. Promega Corporation.