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Reducing the impact of endogenous ribonucleases on reverse transcription-PCR assay systems.

Rapid degradation of RNA is the most important factor impeding the analysis of gene expression in human cells and tissues. Once a tissue has been removed from its normal environment, the relative rates of RNA synthesis and degradation change. This in turn gives rise to changes in the relative proportions of various RNA species within a tissue, as well as to an overall reduction of RNA concentrations.

Several approaches have been used to reduce the rate at which RNA concentrations decline after tissue collection. Contamination of samples with exogenous ribonucleases (RNases), particularly those used in the laboratory during RNase treatment of DNA isolates, is universally recognized as an important cause of RNA loss. The use of meticulous laboratory technique to reduce this contamination is absolutely essential to preserve RNA substrates, but is not sufficient to permit analysis of all RNA targets. Isolation of RNA, along with the use of RNase inhibitors, such as diethyl pyrocarbonate, or chaotropic agents, such as guanidinium chloride or guanidinium isothiocyanate, often used in combination, is also helpful, but also is laborious and time-consuming. Rapid fixation of tissue samples with neutral-buffered formalin or precipitating agents, such as ethanol, destroys RNase activity, but increases the complexity of RNA isolation and may inhibit polymerase activity in reverse transcription (RT)-PCR assays.

To reduce the time required for RNA-based assays (1, 2) and the analytical variation resulting from RNA extraction and purification (3), several investigators have proposed RT-PCR techniques that do not require RNA isolation. The time available for RNA degradation to occur is minimized by the short time (typically -1 h) required by these assays (1). Many laboratories have been unsuccessful in implementing these procedures, however, and assay systems relying on RNA extraction dominate the molecular laboratory medicine community.

In this issue, Hamalainen et al. (4) demonstrate that a potent RNase, eosinophil-derived neurotoxin, is the major factor in reducing the sensitivity of RT-PCR-based assays for minimal residual disease in patients with chronic myelogenous leukemia. Removing eosinophils, either by the use of monoclonal antibodies or by density gradient separation, substantially increases the signal obtained in an RT-PCR assay that does not rely on RNA isolation. The result is an assay that may be quicker, more sensitive, and more reproducible than previous RT-PCR-based assays for the Philadelphia chromosome (bcr/abl translocation) that use either cell lysates or isolated RNA. If this approach can be widely implemented, it represents a substantial advance.

Eosinophil-derived neurotoxin is a member of the RNase A superfamily. This family consists of several closely related nonsecretory RNases coded by genes localized in the q24 to q31 region of human chromosome 14 (5-8). These genes each consist of two exons: a noncoding exon 1 separated by a single intron from the coding sequence in exon 2 (9). The proteins share identical active site residues (10) and nearly identical secondary structures (11). Eosinophil-derived neurotoxin (RNase 2) is expressed only in phagocytic cells, including neutrophils, the large granule of eosinophils, and monocytes (5).

Several similar endogenous, nonsecretory RNases also complicate the molecular analysis of human tissues. RNase 1 is expressed in nearly all healthy human tissues (5). In contrast, a second eosinophil RNase, the eosinophil cationic protein (RNase 3) is found only in eosinophils (5). RNase 4 is found in several human somatic tissues, including liver, pancreas, lung, skeletal muscle, kidney, and placenta, but not in brain (12). This RNase, or an antigenically similar protein, is also found in serum. Angiogenin, a RNase involved in angiogenesis, has a similar tissue distribution (5). RNase 6 is found in healthy human monocytes and neutrophils, but not eosinophils (13). It is likely that all the members of the RNase A superfamily family arose from duplication of a single primordial RNase gene.

In spite of their highly homologous secondary structures and identical key active site amino acids, these RNases differ in function. For example, in addition to its angiogenic effects, angiogenin specifically degrades transfer RNA, but not messenger RNA (14). Eosinophil cationic protein has an RNase activity <1% of that shown by eosinophil-derived neurotoxin (15). Because the relative concentrations of the various RNases vary from tissue to tissue, the effect on any RT-PCR assay of chemically or physically removing any single enzymatic or tissue constituent is difficult to predict.

Hamalainen et al. (4) show that RT-PCR after total RNA extraction yields an assay system detection limit for the bcr/abl translocation of approximately fourfold the theoretical minimum. The physical removal of eosinophils from the RT-PCR assay improves the detection limit by approximately threefold in comparison with total RNA extraction. Density gradient separation using the Ficoll-Hypaque method improves the detection limit approximately twofold. Even after the removal of eosinophils, some RNase activity, presumably arising from RNase 1 and RNases 4-6, remains.

The substantial improvement in the detection limit following removal of eosinophils suggests that molecular diagnosticians should pay greater attention to the cellular and molecular constituents of tissues that are being used in (or contaminating) their assays. Although it may be impossible to remove all endogenous RNases from RTPCR assay systems, in some cases an RNase-rich cellular component (such as eosinophils) may be separated physically from the assay target. In addition to the techniques outlined by Hamalainen et al. (4), microdissection of frozen sections may be helpful for some samples. In other cases, it may be possible to optimize the concentrations of nonspecific RNase inhibitors, such as diethyl pyrocarbonate, by considering the tissues that are represented in the assay system. Finally, it may be possible to design specific RNase inhibitors (such as anti-RNase monoclonal antibodies) that, when added to tissues during or immediately after cell lysis, reduce the effects of RNases more effectively than the nonspecific inhibitors currently in common use. The efforts we take to reduce the influence of internal RNases on RNA analysis may well have even greater payoffs in sensitivity and reproducibility than those that result from the use of meticulous laboratory technique to reduce external contamination.

References

(1.) Klebe RJ, Grant GM, Grant AM, Garcia MA, Giambernardi TA, Taylor GP. RT-PCR without RNA isolation. Biotechniques 1996;21:1094-100.

(2.) Shi YJ, Liu JZ. Direct reverse transcription-polymerase chain reaction from whole blood without RNA extraction. Genet Anal Tech Appl 1992;9:149-50.

(3.) Eskola JU, Hamalainen MM, Nanto V, Rajamaki A, Dahlen P, litia N, Siitari H. Detection of Philadelphia chromosome using PCR and europium-labeled DNA probes. Clin Biochem 1994;27:373-9.

(4.) Hamalainen MM, Eskola JU, Hellman J, Pulkki K. Major interference from leukocytes in reverse transcription-PCR identified as neurotoxin ribonuclease from eosinophils: detection of residual chronic myelogenous leukemia from cell lysates by use of an eosinophil-depleted cell preparation. Clin Chem 1999;45:465-71.

(5.) Futami J, Tsushima Y, Murato Y, Tada H, Sasaki J, Seno M, Yamada H. Tissue-specific expression of pancreatic-type RNases and RNase inhibitor in humans. DNA Cell Biol 1997;16:413-9.

(6.) Hamann KJ, Ten RM, Loegering DA, Jenkins RB, Heise MT, Schad CR, et al. Structure and chromosome localization of the human eosinophil-derived neurotoxin and eosinophil cationic protein genes: evidence for intronless coding sequences in the ribonuclease gene superfamily. Genomics 1990; 7:535-46.

(7.) Mastrianni DM, Eddy RL, Rosenberg HF, Corrette SE, Shows TB, Tenen DG, Ackerman SJ. Localization of the human eosinophil Charcot-Leyden crystal protein (lysophospholipase) gene (CLC) to chromosome 19 and the human ribonuclease 2 (eosinophil-derived neurotoxin) and ribonuclease 3 (eosinophil cationic protein) genes (RNS2and RNS3) to chromosome 14. Genomics 1992;13:240-2.

(8.) Rosenberg HF, Dyer KID. Diversity among the primate eosinophil-derived neurotoxin genes: a specific C-terminal sequence is necessary for enhanced ribonuclease activity. Nucleic Acids Res 1997;25:3532-6.

(9.) Tiffany HL, Handen JS, Rosenberg HF. Enhanced expression of the eosinophil-derived neurotoxin ribonuclease (RNS2) gene requires interaction between the promoter and intron. J Biol Chem 1996;271:12387-93.

(10.) Rosenberg HF, Ackerman SJ, Tenen DG. Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity. J Exp Med 1989;170:163-76.

(11.) Mosimann SC, Newton DL, Youle RJ, James MN. X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin at 1.83 A resolution. J Mol Biol 1996;260:540-52.

(12.) Rosenberg HF, Dyer KID. Human ribonuclease 4 (RNase 4): coding sequence, chromosomal localization and identification of two distinct transcripts in human somatic tissues. Nucleic Acids Res 1995;23:4290-5.

(13.) Rosenberg HF, Dyer KID. Molecular cloning and characterization of a novel human ribonuclease (RNase k6): increasing diversity in the enlarging ribonuclease gene family. Nucleic Acids Res 1996;24:3507-13.

(14.) Saxena SK, Rybak SM, Davey RT Jr, Youle RJ, Ackerman EJ. Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily. J Biol Chem 1992;267:21982-6.

(15.) Gullberg U, Widegren B, Amason U, Egesten A, Olsson I. The cytotoxic eosinophil cationic protein (ECP) has ribonuclease activity. Biochem Biophys Res Commun 1986;139:1239-42.

Timothy J. O'Leary

Department of Cellular Pathology

Armed Forces Institute of Pathology

Washington, DC 20306-6000

Fax 202-782-7623

E-mail oleary@afip.osd.mil
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Title Annotation:Editorial
Author:O'Leary, Timothy J.
Publication:Clinical Chemistry
Date:Apr 1, 1999
Words:1478
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