This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kopreski, M. S. Articles by Gocke, C. D. Search for Related Content PubMed PubMed Citation Articles by Kopreski, M. S. Articles by Gocke, C. D.

Detection of Tumor Messenger RNA in the Serum of Patients with Malignant Melanoma

OncoMEDx, Inc., Columbia, Maryland [M. S. K., C. D. G.]; National Cancer Institute, Bethesda, Maryland [L. W. K.]; and Penn State Geisinger-Hershey Medical Center, Hershey, Pennsylvania [F. A. B., C. D. G.]

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Serum RNases are known to be elevated in patients with cancer. Consequently, it is not clear whether human mRNA with sufficient integrity as to permit reverse transcription-PCR (RT-PCR) amplification is detectable in serum. We examined serum from six patients with malignant melanoma for human tyrosinase mRNA using RT-PCR. Serum from 20 normal volunteers served as controls. Tyrosinase mRNA could be demonstrated in serum from four of the six melanoma patients with detection by gel electrophoresis and confirmation by blotting amplified product to a tyrosinase-specific probe. The serum remained tyrosinase mRNA positive, even if passed through a 0.45 µm filter prior to RNA extraction, indicating that the mRNA was extracellular at the time of extraction. Tyrosinase mRNA could not be detected in any control serum (0 of 20 individuals). The presence and integrity of amplifiable RNA was confirmed in all serum specimens (patients and controls) by RT-PCR amplification of c-abl mRNA. Amplifiable RNA could be demonstrated regardless of whether serum was freshly drawn or stored frozen for several years. We conclude that human mRNA can be extracted and amplified from serum. The ability to amplify tumor mRNA from serum may have important utility in cancer diagnostics and monitoring.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
The development of sensitive RNA-based amplification methods has enabled new approaches to diagnosing and monitoring malignancy. One approach under investigation detects circulating cancer cells by extracting and amplifying specific tumor RNA from the cellular fraction of peripheral blood (1 , 2) . Commonly, cellular tyrosinase mRNA has been assayed in amplification strategies to detect circulating malignant melanoma cells (3 , 4) . However, circulating cancer cells tend to correlate with tumor burden, with lower rates of detection seen in those with minimal or early disease (5) . The detection of circulating melanoma cells has similarly been shown to correlate with the stage of disease, with low detection rates seen in localized disease (6, 7, 8) . More sensitive approaches are needed to monitor patients with low tumor burden.

Recently, it has been shown that amplifiable extracellular tumor DNA can be found in plasma and serum (9, 10, 11, 12, 13, 14, 15) . The extracellular DNA appears detectable, even in the absence of circulating cancer cells (10 , 11) , and can be detected in those with early disease and low tumor burden (16 , 17) . However, mRNA is more fragile than DNA and is presumed to be highly susceptible to degradation by blood RNases. Furthermore, blood RNases are known to increase in patients with cancer (18) . It has thus been commonly presumed that amplifiable human RNA could not survive extracellularly in bodily fluids. Although several reports have suggested that RNA might be present in plasma or serum (19, 20, 21, 22) , it remained to be shown whether extracellular RNA exists with sufficient integrity as to allow RT-PCR² amplification. In this preliminary study, we evaluate whether amplifiable tyrosinase mRNA is detectable in serum from patients with malignant melanoma.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Serum Preparation.
Serum from six patients with metastatic malignant melanoma treated on clinical protocols of the former Biological Response Modifiers Program, National Cancer Institute (Frederick, MD), and serum from 20 normal volunteer controls (rendered anonymous) were assayed. Initially, the sera from melanoma patients were prepared by centrifugation of clotted peripheral blood at 830 x g for 10 min, followed by careful aliquoting and freezing of the serum. Although patient serum specimens were generally prepared and frozen within 4 h of the blood draw, preparation time varied. All melanoma patient sera were initially frozen at -70°C and stored for >2 years prior to use. Control serum was prospectively obtained and prepared in a similar fashion, except that to minimize mRNA degradation, which we found to be associated with freeze-thaw cycles, control serum was not frozen prior to RNA extraction, which was also generally performed within 4 h of blood draw.

RNA was extracted from 50 µl of serum using a commercial kit, Perfect RNA: Total RNA Isolation kit (5 Prime-3 Prime, Inc., Boulder, Colorado), performed according to the manufacturer’s direction. The concentration of RNA was then approximated by spectrophotometry.

Amplification.
Comparable quantities of RNA per serum aliquot corresponding to 10–45% of the RNA extracted from 50 µl of serum were reverse transcribed by a method adapted from Kawasaki (23) . A mixture was prepared consisting of the RNA, 3 µl of 0.1 M DTT, 3 µl of x10 Taq buffer (Fisher, Pittsburgh, PA), 100 pmol of random hexamer primers (Promega Corp., Madison, WI), 3 µl of deoxynucleotides (10 mM each; New England BioLabs, Beverly, MA), 4.8 µl (25 mM) magnesium chloride (Fisher), 1 µl RNAsin (25 units/µl; Promega), 3 µl of AMV reverse transcriptase (3 units/µl; Promega) diluted 1:10 in water, for a total volume of 30 µl. This was incubated at room temperature for 10 min and then maintained in a heat block at 42°C for 60 min. Fifteen µl of cDNA were then used in the amplification reaction.

Human tyrosinase cDNA was amplified by nested PCR as adapted from Smith et al. (3) . Tyrosinase primers were: TYR 1 (outer, sense), 5'-TTGGCAGATTGTCTGTAGCC; TYR 2 (outer, antisense), 5'-AGGCATTGTGCATGCTGCTT; TYR 3 (nested, sense), 5'-GTCTTTATGCAATGGAACGC; and TYR 4 (nested, antisense), 5'-GCTATCCCAGTAAGTGGACT.

A reaction mixture was prepared consisting of 1 x reaction buffer (Fisher), 1.6 mM magnesium chloride (Fisher), 200 µM each dATP, dCTP, dGTP, and dTTP (New England BioLabs), 2.5 pmol each of primer TYR-1 and TYR-2, 1 unit Taq polymerase (Fisher), and distilled water for a total volume of 50 µl. This reaction mixture was overlaid with mineral oil and amplified for 15 cycles in a Ericomp Delta Cycler 1, with denaturation at 94°C for 1 min, annealing at 53°C for 1 min, and extension at 72°C for 1 min. Reamplification with nested primers TYR 3 and TYR 4 was then performed, in which the first amplification product was diluted 1:100 in distilled water, with 5 µl of dilution used as template for the second amplification step. The reaction mixture was identical to that in the first amplification step except that primers consisted of 25 pmol each of TYR 3 and TYR 4. This mixture was cycled for 40 cycles at the same parameters, with a final extension at 72°C for 8 min.

To verify the presence and integrity of serum RNA, all sera were additionally assayed for human c-abl mRNA, a mRNA expected to be expressed in all individuals. RT-PCR for c-abl was performed using 15 µl of the cDNA in a reaction mixture with 1 x PCR buffer, 1.5 mM magnesium chloride, 200 µM each deoxynucleotide triphosphates, 50 pmol of Abl-1 primer (Oncogene Science, Uniondale, NY), 50 pmol of Abl-3 primer (Oncogene Science), and 2.5 units of Taq polymerase in a 50-µl volume. This mixture was cycled 35 times with an initial denaturing temperature of 94°C for 4 min, followed by a denaturing temperature of 94°C for 60 s, annealing temperature of 65°C for 60 s, and extension temperature of 72°C for 90 s. A nested PCR reaction was then prepared using the same constituents as above, except that 4 µl of the first-round product was used in place of the 15 µl of cDNA, the water was reduced by 1 µl, and primers used were Abl-2 and Abl-4 (Oncogene Science). The mixture was then cycled 35 times under the same cycling parameters (manufacturer’s Abl protocol, CML primer set; Oncogene Science).

All RT-PCR amplifications were performed with particular attention paid to prevention of contamination. Amplifications included a tyrosinase-positive control consisting of tyrosinase-positive melanoma tissue and a negative control lacking cDNA. The risk of contamination yielding falsely positive results was further minimized by repeating the assay on the melanoma patient sera using additional, separately prepared serum aliquots.

Detection.
Amplified product was electrophoresed through a 4% agarose gel (2:1 NuSieve GTG; FMC Bioproducts, Rockland, Maine) in 1x TBE buffer (pH 8.0) at 100 V for 2 h and stained with ethidium bromide. The nested tyrosinase primers amplify a PCR product of 207 bp. The c-abl mRNA yields an amplified product 267 bp long.

To further verify tyrosinase results, PCR products from the gel were transferred by Southern blot onto a nylon membrane (MSI, Westboro, MA). Replicate blots were then probed by TYR-5, a tyrosinase internal probe (bp 920–934 of cDNA; 5'-CCAGAACCCCAAGGC). Hybridization and wash conditions were as specified by the membrane’s manufacturer.

RNA Stability and Abundance.
To evaluate whether RNA extracted from serum was extracellular at the time of extraction, sera from two melanoma patients were reassayed by first passing the thawed centrifuged serum through a 0.45 µm cellulose acetate filter (Nalgene, Rochester, NY) prior to RNA extraction. Reverse transcription, amplification, and detection of both tyrosinase and c-abl mRNA were then carried out as described previously.

The stability of the serum RNA under different conditions was evaluated. To test the stability through several freeze-thaw cycles, identical aliquots of serum were repeatedly frozen to -20°C and rapidly thawed prior to extracting RNA as described above. To determine the rapidity of degradation of the RNA, thawed serum was placed at 4°C with aliquots drawn off for extraction at 15-min intervals. The effect of adding RNase inhibitors to serum prior to freezing was assayed by adding 25 units of RNAsin (Promega) or 50 µl of 2 M guanidinium thiocyanate (United States Biochemical Corp., Cleveland, OH) to 50-µl aliquots of serum. Finally, a semiquantitative assessment of RNA abundance was made by preparing 10-fold dilutions of both freshly obtained (three patients) and frozen-thawed serum (five patients), extracting, and testing for c-abl mRNA and (in one melanoma patient serum) tyrosinase mRNA.

	Results

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Serial dilutions into water of RNA extracted from melanoma tissue demonstrated the ability of the assay to detect tyrosinase mRNA in a 0.4-pg sample (Fig. 1) . c-abl mRNA could be demonstrated in the serum of all patients and all normal volunteers, confirming both the presence and integrity of RNA in serum (Figs. 1 and 2 ). Tyrosinase mRNA was detected in serum from four of the six (67%) malignant melanoma patients assayed (Fig. 1) . Results were reproducible upon retesting of additional serum aliquots. In contrast, none of the 20 normal control sera tested positive for tyrosinase mRNA (Fig. 2A) . In all amplification assays, the tyrosinase-positive melanoma tissue and cDNA-absent sample tested appropriately. Southern blotting of the amplified product confirmed detection of tyrosinase cDNA in all four gel-positive melanoma patients (Fig. 1B) .

View larger version (62K): [in this window] [in a new window]   Fig. 1. Tyrosinase mRNA in melanoma patient serum. A, RT-PCR of serum from melanoma patients and positive controls using nested tyrosinase primers yields the expected 207-bp product. B, Southern blot with internal probe TYR-5. C, RT-PCR of serum from melanoma patients using nested c-abl primers. Lane 1, 4 pg of RNA extracted from melanoma tissue; Lane 2, 0.4 pg; Lane 3, 0.04 pg; Lanes 4–9, sera from six melanoma patients; Lane -, water blank.

View larger version (72K): [in this window] [in a new window]   Fig. 2. Absence of tyrosinase mRNA in normal control sera. A, tyrosinase RT-PCR of control sera. B, c-abl RT-PCR. Lane 1, 4 pg of RNA extracted from melanoma tissue; Lane 2, 0.4 pg; Lanes 3–12, sera from 10 normal donors; Lane -, water blank.

View larger version (29K): [in this window] [in a new window]   Fig. 3. The filtering of serum does not eliminate tyrosinase mRNA. Sera from two patients (Lanes 1 and 2) were assayed by RT-PCR for tyrosinase mRNA before (left panel) and after (right panel) passage through a 0.45 µm filter. Lane -, water blank.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Serum mRNA stability studies. A, time course experiment. Aliquots of a melanoma patient’s serum were thawed and incubated on ice for 0, 15, 30, or 60 min, followed by RNA extraction and RT-PCR for tyrosinase mRNA. Lane 1, 4 pg of RNA extracted from melanoma tissue; Lane 2, 0.4 pg. Lane -, water blank. B, effect of freeze-thaw cycles on signal. Tyrosinase RT-PCR of melanoma patient serum extracted after two freeze-thaw cycles (Lane 3), three freeze-thaw cycles (Lane 4), four freeze-thaw cycles (Lane 5), and five freeze-thaw cycles (Lane 6) is shown. Other lanes as above. C, PCR for c-abl mRNA from three never-frozen normal control sera, each serially diluted 10-fold after reverse transcription. D, PCR for c-abl mRNA from the same sera, frozen and thawed once, then serially diluted after reverse transcription. Lane -, water blank.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Relative abundance of tyrosinase and c-abl mRNA in serum from a patient with melanoma. cDNA was serially diluted 10-fold in water (dilution indicated above each lane) and then assayed in parallel for tyrosinase or c-abl. Lane -, water blank.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

In this study, we demonstrate that amplifiable human mRNA is present in serum. Human tyrosinase mRNA was detected in the serum of four of six patients with malignant melanoma. In addition, c-abl mRNA was detected in the serum of all individuals tested. The RNA was detectable, even when centrifuged serum was passed through a filter, indicating that the mRNA was extracellular at the time of extraction from serum. Of interest, tyrosinase mRNA was detectable in serum, although the serum had been stored frozen over several years, suggesting that stored serum banks may be used in RT-PCR-based investigations, although the integrity of the RNA is affected during multiple freeze-thaw cycles as noted. We have also found that human mRNA may be amplified from plasma (results not shown).

At the present time, it is not known how serum RNA is protected from blood RNases. It is possible that extracellular mRNA could circulate bound to protein or phospholipid, thus being protected from nucleases. RNA has been found on the cell surface of cancer cells (25 , 26) and could be shed within vesicles (27 , 28) . Rosi et al. (29) , using nuclear magnetic resonance spectroscopy, described RNA-lipid vesicles shed in vitro from a human colon adenocarcinoma cell line. Further characterization of the RNA-lipid vesicles indicated the presence of triglycerides, cholesterol esters, lipids, oligopeptides, and phospholipids (30) . Mountford et al. (20) identified a similar proteolipid in the plasma of a patient with an ovarian neoplasm using magnetic resonance spectroscopy. Evaluation of the proteolipid with the orcinol method suggested RNA to be present, although this could not be confirmed using other methods. Furthermore, Stroun et al. (31) found that RNA-DNA nucleoprotein complexes were actively released from normal cells in culture, and Wieczorek et al. (21 , 22) described a RNA-proteolipid complex in the sera of cancer patients that appeared to be an actively secreted product of tumor cells. It is not known whether extracellular tyrosinase mRNA circulates within analogous RNA-proteolipid complexes. RNA could similarly circulate within apoptotic bodies or nuclear fragments. Nuclear RNA-protein complexes, possibly representing functional nuclear suborganellular elements, have been described (32) . Alternatively, it is possible that clearance of free RNA in vivo by RNase is not as rapid as believed previously. Free RNA could potentially be released after in vivo or in vitro lysis of tumor cells found in the blood. Further investigation is needed to clarify the etiological and pathophysiological nature of extracellular RNA. Within this context, one may consider the effect that one or more freeze-thaw cycles have upon extracellular RNA stability. In addition to prolonging the potential exposure of RNA to nucleases, it is possible that during the freeze-thaw process, vesicles or apoptotic bodies are disrupted or protein-RNA interaction otherwise affected, thereby rendering the RNA more susceptible to nucleases.

Although the observations presented in this study must be considered preliminary, the finding that tumor mRNA is amplifiable from serum may offer a new approach to cancer diagnostics, monitoring, and pharmacogenomic evaluation. Similar to tyrosinase mRNA, other tumor mRNA should be demonstrable in serum and plasma in other malignancies. One could further anticipate finding extracellular RNA in other bodily fluids. The present study was limited to patients with metastatic cancer. Whether serum or plasma-based tumor mRNA assays will prove sensitive in diagnosing and monitoring early or limited disease remains to be clarified. However, the demonstration that amplifiable tumor mRNA is present in serum offers a new avenue of exploration. Future clinical trials are needed to address the potential of this approach.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Pharmacia & Upjohn, 1015-298-163, 7000 Portage Road, Kalamazoo, MI 49001. Phone: (616) 833-0980; Fax: (616) 833-1476.

² The abbreviation used is: RT-PCR, reverse transcription-PCR.

Received 12/16/98; revised 4/26/99; accepted 4/27/99.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

1. Datta Y. H., Adams P. T., Drobyski W. R., Ethier S. P., Terry V. H., Roth M. S. Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction. J. Clin. Oncol., 12: 475-482, 1994.[Abstract]
2. Moreno J. G., Croce C. M., Fischer R., Monne M., Vihko P., Mulholland S. G., Gomella L. G. Detection of hematogenous micrometastasis in patients with prostate cancer. Cancer Res., 52: 6110-6112, 1992.[Abstract/Free Full Text]
3. Smith B., Selby P., Southgate J., Pittman K., Bradley C., Blair G. E. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet, 338: 1227-1229, 1991.[Medline]
4. Brossart P., Keilholz U., Scheibenbogen C., Mohler T., Willhauck M., Hunstein W. Detection of residual tumor cells in patients with malignant melanoma responding to immunotherapy. J. Immunother., 15: 38-41, 1994.
5. Hardingham J. E., Kotasek D., Sage R. E., Eaton M. C., Pascoe V. H., Dobrovic A. Detection of circulating tumor cells in colorectal cancer by immunobead-PCR is a sensitive prognostic marker for relapse of disease. Mol. Med., 1: 789-794, 1995.[Medline]
6. Glaser R., Rass K., Seiter S., Hauschild A., Christophers E., Tilgen W. Detection of circulating melanoma cells by specific amplification of tyrosinase complementary DNA is not a reliable tumor marker in melanoma patients: a clinical two-center study. J. Clin. Oncol., 15: 2818-2825, 1997.[Abstract]
7. Farthmann B., Eberle J., Krasagakis K., Gstottner M., Wang N., Bisson S., Orfanos C. E. RT-PCR for tyrosinase-mRNA-positive cells in peripheral blood: evaluation strategy and correlation with known prognostic markers in 123 melanoma patients. J. Invest. Dermatol., 110: 263-267, 1998.[Medline]
8. Reinhold U., Ludtke-Handjery H. C., Schnautz S., Kreysel H. W., Abken H. The analysis of tyrosinase-specific mRNA in blood samples of melanoma patients by RT-PCR is not a useful test for metastatic tumor progression. J. Invest. Dermatol., 108: 166-169, 1997.[Medline]
9. Sorenson G. D., Pribish D. M., Valone F. H., Memoli V. A., Bzik D. J., Yao S-L. Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol. Biomark. Prev., 3: 67-71, 1994.[Abstract]
10. Kopreski M. S., Benko F. A., Kwee C., Leitzel K. E., Eskander E., Lipton A., Gocke C. D. Detection of mutant K-ras DNA in plasma or serum of patients with colorectal cancer. Br. J. Cancer, 76: 1293-1299, 1997.[Medline]
11. Vasioukhin V., Anker P., Maurice P., Lyautey J., Lederrey C., Stroun M. Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia. Br. J. Haematol., 86: 774-779, 1994.[Medline]
12. Anker P., Lefort F., Vasioukhin V., Lyautey J., Lederrey C., Chen X. Q., Stroun M., Mulcahy H. E., Farthing M. J. G. K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer. Gastroenterology, 112: 1114-1120, 1997.[Medline]
13. Chen X. Q., Stroun M., Magnenat J-L., Nicod L. P., Kurt A-M., Lyautey J., Lederrey C., Anker P. Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat. Med., 9: 1033-1035, 1996.
14. Nawroz H., Koch W., Anker P., Stroun M., Sidransky D. Microsatellite alterations in serum DNA of head and neck cancer patients. Nat. Med., 9: 1035-1037, 1996.
15. Hibi K., Robinson C. R., Booker S., Wu L., Hamilton S. R., Sidransky D., Jen J. Molecular detection of genetic alterations in the serum of colorectal cancer patients. Cancer Res., 58: 1405-1407, 1998.[Abstract/Free Full Text]
16. Kopreski M. S., Benko F. A., Borys D. J., Khan A., Kwee C. M., McGarrity T. J., Gocke C. D. Mutated KRAS DNA in plasma as a marker in early colorectal neoplasia. Proc. Am. Soc. Clin. Oncol., 17: 269 1998.
17. Mulcahy H. E., Lyautey J., Lederrey C., Chen X. Q., Anker P., Alstead E. M., Ballinger A., Farthing M. J. G., Stroun M. A prospective study of K-ras mutations in the plasma of pancreatic cancer patients. Clin. Cancer Res., 4: 271-275, 1998.[Abstract]
18. Reddi K. K., Holland J. F. Elevated serum ribonuclease in patients with pancreatic cancer. Proc. Natl. Acad. Sci. USA, 73: 2308-2310, 1976.[Abstract/Free Full Text]
19. Kamm R. C., Smith A. G. Nucleic acid concentrations in normal human plasma. Clin. Chem., 18: 519-522, 1972.[Abstract]
20. Mountford C. E., May G. L., Wright L. C., MacKinnon W. B., Dyne M., Holmes K. T., Van Haaften-Day C., Tattersall M. H. N. Proteolipid identified by magnetic resonance spectroscopy in plasma of a patient with borderline ovarian tumour. Lancet, 1: 829-834, 1987.[Medline]
21. Wieczorek A. J., Rhyner C., Block L. H. Isolation and characterization of an RNA-proteolipid complex associated with the malignant state in humans. Proc. Natl. Acad. Sci. USA, 82: 3455-3459, 1985.[Abstract/Free Full Text]
22. Wieczorek A. J., Sitaramam V., Machleidt W., Rhyner K., Perruchoud A. P., Block L. H. Diagnostic and prognostic value of RNA-proteolipid in sera of patients with malignant disorders following therapy: first clinical evaluation of a novel tumor marker. Cancer Res., 47: 6407-6412, 1987.[Abstract/Free Full Text]
23. Kawasaki E. S. Amplification of RNA Innis M. A. Gelfand D. H. Sninsky J. J. White T. J. eds. . PCR Protocols: A Guide to Methods and Applications, : 23-27, Academic Press San Diego 1990.
24. Komeda T., Fukuda Y., Sando T., Kita R., Furukawa M., Nishida N., Amenomori M., Nakao K. Sensitive detection of circulating hepatocellular carcinoma cells in peripheral venous blood. Cancer (Phila.), 75: 2214-2219, 1995.[Medline]
25. Juckett D. A., Rosenberg B. Actions of cis-diamminedichloroplatinum on cell surface nucleic acids in cancer cells as determined by cell electrophoresis techniques. Cancer Res., 42: 3565-3573, 1982.[Abstract/Free Full Text]
26. Rieber M., Bacalao J. An "external" RNA removable from mammalian cells by mild proteolysis. Proc. Natl. Acad. Sci. USA, 71: 4960-4964, 1974.[Abstract/Free Full Text]
27. Barz D., Goppelt M., Szamel M., Schirrmacher V., Resch K. Characterization of cellular and extracellular plasma membrane vesicles from a non-metastasing lymphoma (Eb) and its metastasing variant (Esb). Biochim. Biophys. Acta, 814: 77-84, 1985.[Medline]
28. Carr J. M., Dvorak A. M., Dvorak H. F. Circulating membrane vesicles in leukemic blood. Cancer Res., 45: 5944-5951, 1985.
29. Rosi A., Guidoni L., Luciani A. M., Mariutti G., Viti V. RNA-lipid complexes released from the plasma membrane of human colon carcinoma cells. Cancer Lett., 39: 153-160, 1988.[Medline]
30. Masella R., Cantafora A., Guidoni L., Luciani A. M., Mariutti G., Rosi A., Viti V. Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells. FEBS Lett., 246: 25-29, 1989.[Medline]
31. Stroun M., Anker P., Beljanski M., Henri J., Lederrey C., Ojha M., Maurice P. A. Presence of RNA in the nucleoprotein complex spontaneously released by human lymphocytes and frog auricles in culture. Cancer Res., 38: 3546-3554, 1978.[Abstract/Free Full Text]
32. Rosenberg-Nicolson N. L., Nicolson G. L. Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities. J. Cell. Biochem., 50: 43-52, 1992.[Medline]

This article has been cited by other articles:

				J. M. Garcia, V. Garcia, C. Pena, G. Dominguez, J. Silva, R. Diaz, P. Espinosa, M. J. Citores, M. Collado, and F. Bonilla Extracellular plasma RNA from colon cancer patients is confined in a vesicle-like structure and is mRNA-enriched RNA, July 1, 2008; 14(7): 1424 - 1432. [Abstract] [Full Text] [PDF]

				J. M. Garcia, C. Pena, V. Garcia, G. Dominguez, C. Munoz, J. Silva, I. Millan, R. Diaz, Y. Lorenzo, R. Rodriguez, et al. Prognostic Value of LISCH7 mRNA in Plasma and Tumor of Colon Cancer Patients Clin. Cancer Res., November 1, 2007; 13(21): 6351 - 6358. [Abstract] [Full Text] [PDF]

				S. Rani, M. Clynes, and L. O'Driscoll Detection of Amplifiable mRNA Extracellular to Insulin-Producing Cells: Potential for Predicting Beta Cell Mass and Function Clin. Chem., November 1, 2007; 53(11): 1936 - 1944. [Abstract] [Full Text] [PDF]

				M. Collado, V. Garcia, J. M. Garcia, I. Alonso, L. Lombardia, R. Diaz-Uriarte, L. A. Lopez Fernandez, A. Zaballos, F. Bonilla, and M. Serrano Genomic Profiling of Circulating Plasma RNA for the Analysis of Cancer Clin. Chem., October 1, 2007; 53(10): 1860 - 1863. [Abstract] [Full Text] [PDF]

				S. Fischer, T. Gerriets, C. Wessels, M. Walberer, S. Kostin, E. Stolz, K. Zheleva, A. Hocke, S. Hippenstiel, and K. T. Preissner Extracellular RNA mediates endothelial-cell permeability via vascular endothelial growth factor Blood, October 1, 2007; 110(7): 2457 - 2465. [Abstract] [Full Text] [PDF]

				B. G ZIMMERMANN, N. J. PARK, and D. T WONG Genomic Targets in Saliva Ann. N.Y. Acad. Sci., March 1, 2007; 1098(1): 184 - 191. [Abstract] [Full Text] [PDF]

				M. FLEISCHHACKER Biology of Circulating mRNA: Still More Questions Than Answers? Ann. N.Y. Acad. Sci., September 1, 2006; 1075: 40 - 49. [Abstract] [Full Text] [PDF]

				K. BOTTCHER, A. WENZEL, and J. M WARNECKE Investigation of the Origin of Extracellular RNA in Human Cell Culture. Ann. N.Y. Acad. Sci., September 1, 2006; 1075: 50 - 56. [Abstract] [Full Text] [PDF]

				J. SAMOS, D. C GARCIA-OLMO, M. G PICAZO, A. RUBIO-VITALLER, and D. GARCIA-OLMO Circulating Nucleic Acids in Plasma/Serum and Tumor Progression: Are Apoptotic Bodies Involved? An Experimental Study in a Rat Cancer Model. Ann. N.Y. Acad. Sci., September 1, 2006; 1075: 165 - 173. [Abstract] [Full Text] [PDF]

				B. C.K WONG and Y.M DENNIS LO Plasma RNA Integrity Analysis: Methodology and Validation. Ann. N.Y. Acad. Sci., September 1, 2006; 1075: 174 - 178. [Abstract] [Full Text] [PDF]

				F DASI, P MARTINEZ-RODES, J.A MARCH, J SANTAMARIA, J.M MARTINEZ-JAVALOYAS, M GIL, and S.F ALINO Real-Time Quantification of Human Telomerase Reverse Transcriptase mRNA in the Plasma of Patients with Prostate Cancer. Ann. N.Y. Acad. Sci., September 1, 2006; 1075: 204 - 210. [Abstract] [Full Text] [PDF]

				E. PAPADOPOULOU, E. DAVILAS, V. SOTIRIOU, E. GEORGAKOPOULOS, S. GEORGAKOPOULOU, A. KOLIOPANOS, F. AGGELAKIS, K. DARDOUFAS, N. J AGNANTI, I. KARYDAS, et al. Cell-free DNA and RNA in Plasma as a New Molecular Marker for Prostate and Breast Cancer. Ann. N.Y. Acad. Sci., September 1, 2006; 1075: 235 - 243. [Abstract] [Full Text] [PDF]

				C. DING and Y. M. D. LO MALDI-TOF Mass Spectrometry for Quantitative, Specific, and Sensitive Analysis of DNA and RNA. Ann. N.Y. Acad. Sci., September 1, 2006; 1075: 282 - 287. [Abstract] [Full Text] [PDF]

				E. Y RYKOVA, W. WUNSCHE, O. E BRIZGUNOVA, T. E SKVORTSOVA, S. N TAMKOVICH, I. S SENIN, P. P LAKTIONOV, G. SCZAKIEL, and V. V VLASSOV Concentrations of Circulating RNA from Healthy Donors and Cancer Patients Estimated by Different Methods. Ann. N.Y. Acad. Sci., September 1, 2006; 1075: 328 - 333. [Abstract] [Full Text] [PDF]

				B. C.K. Wong, K.C. A. Chan, A. T.C. Chan, S.-F. Leung, L. Y.S. Chan, K. C.K. Chow, and Y.M. D. Lo Reduced Plasma RNA Integrity in Nasopharyngeal Carcinoma Patients Clin. Cancer Res., April 15, 2006; 12(8): 2512 - 2516. [Abstract] [Full Text] [PDF]

				Y. Li, D. Elashoff, M. Oh, U. Sinha, M. A.R. St John, X. Zhou, E. Abemayor, and D. T. Wong Serum Circulating Human mRNA Profiling and Its Utility for Oral Cancer Detection J. Clin. Oncol., April 10, 2006; 24(11): 1754 - 1760. [Abstract] [Full Text] [PDF]

				V. Garcia, J. M. Garcia, C. Pena, J. Silva, G. Dominguez, A. Hurtado, I. Alonso, R. Rodriguez, M. Provencio, and F. Bonilla Thymidylate synthase messenger RNA expression in plasma from patients with colon cancer: prognostic potential. Clin. Cancer Res., April 1, 2006; 12(7): 2095 - 2100. [Abstract] [Full Text] [PDF]

				B. C.K. Wong, R. W.K. Chiu, N. B.Y. Tsui, K.C. A. Chan, L. W. Chan, T. K. Lau, T. N. Leung, and Y.M. D. Lo Circulating Placental RNA in Maternal Plasma Is Associated with a Preponderance of 5' mRNA Fragments: Implications for Noninvasive Prenatal Diagnosis and Monitoring Clin. Chem., October 1, 2005; 51(10): 1786 - 1795. [Abstract] [Full Text] [PDF]

				P. B. Larrabee, K. L. Johnson, I. Peter, and D. W. Bianchi Presence of Filterable and Nonfilterable Cell-Free mRNA in Amniotic Fluid Clin. Chem., June 1, 2005; 51(6): 1024 - 1026. [Full Text] [PDF]

				N. Miura, Y. Maeda, T. Kanbe, H. Yazama, Y. Takeda, R. Sato, T. Tsukamoto, E. Sato, A. Marumoto, T. Harada, et al. Serum Human Telomerase Reverse Transcriptase Messenger RNA as a Novel Tumor Marker for Hepatocellular Carcinoma Clin. Cancer Res., May 1, 2005; 11(9): 3205 - 3209. [Abstract] [Full Text] [PDF]

				Y.M. D. Lo Recent Advances in Fetal Nucleic Acids in Maternal Plasma J. Histochem. Cytochem., March 1, 2005; 53(3): 293 - 296. [Abstract] [Full Text] [PDF]

				F. Z. Bischoff, D. E. Lewis, and J. L. Simpson Cell-free fetal DNA in maternal blood: kinetics, source and structure Hum. Reprod. Update, January 1, 2005; 11(1): 59 - 67. [Abstract] [Full Text] [PDF]

				A M Gilbey, D Burnett, R E Coleman, and I Holen The detection of circulating breast cancer cells in blood J. Clin. Pathol., September 1, 2004; 57(9): 903 - 911. [Abstract] [Full Text] [PDF]

				S C C Wong, E S F Lo, and M T Cheung An optimised protocol for the extraction of non-viral mRNA from human plasma frozen for three years J. Clin. Pathol., July 1, 2004; 57(7): 766 - 768. [Abstract] [Full Text] [PDF]

				Y M D. LO and R. W.K. CHIU The Biology and Diagnostic Applications of Plasma RNA Ann. N.Y. Acad. Sci., June 1, 2004; 1022(1): 135 - 139. [Abstract] [Full Text] [PDF]

				E ENGEL, B SCHMIDT, T CARSTENSEN, S WEICKMANN, B JANDRIG, C WITT, and M FLEISCHHACKER Detection of Tumor-Specific mRNA in Cell-Free Bronchial Lavage Supernatant in Patients with Lung Cancer Ann. N.Y. Acad. Sci., June 1, 2004; 1022(1): 140 - 146. [Abstract] [Full Text] [PDF]

				P. P. LAKTIONOV, S. N. TAMKOVICH, E. YU. RYKOVA, O. E. BRYZGUNOVA, A. V. STARIKOV, N. P. KUZNETSOVA, and V. V. VLASSOV Cell-Surface-Bound Nucleic Acids: Free and Cell-Surface-Bound Nucleic Acids in Blood of Healthy Donors and Breast Cancer Patients Ann. N.Y. Acad. Sci., June 1, 2004; 1022(1): 221 - 227. [Abstract] [Full Text] [PDF]

				Y. Li, X. Zhou, M.A.R. St. John, and D.T.W. Wong RNA Profiling of Cell-free Saliva Using Microarray Technology J. Dent. Res., March 1, 2004; 83(3): 199 - 203. [Abstract] [Full Text]

				U. Keilholz, P. Goldin-Lang, N. E. Bechrakis, N. Max, A. Letsch, A. Schmittel, C. Scheibenbogen, K. Heufelder, A. Eggermont, and E. Thiel Quantitative Detection of Circulating Tumor Cells in Cutaneous and Ocular Melanoma and Quality Assessment by Real-Time Reverse Transcriptase-Polymerase Chain Reaction Clin. Cancer Res., March 1, 2004; 10(5): 1605 - 1612. [Abstract] [Full Text] [PDF]

				S. C. C. Wong, S. F. E. Lo, M. T. Cheung, K. O. E. Ng, C. W. Tse, B. S. P. Lai, K. C. Lee, and Y. M. D. Lo Quantification of Plasma {beta}-Catenin mRNA in Colorectal Cancer and Adenoma Patients Clin. Cancer Res., March 1, 2004; 10(5): 1613 - 1617. [Abstract] [Full Text] [PDF]

				T. El-Hefnawy, S. Raja, L. Kelly, W. L. Bigbee, J. M. Kirkwood, J. D. Luketich, and T. E. Godfrey Characterization of Amplifiable, Circulating RNA in Plasma and Its Potential as a Tool for Cancer Diagnostics Clin. Chem., March 1, 2004; 50(3): 564 - 573. [Abstract] [Full Text] [PDF]

				T. H. Rainer, N. Y.L. Lam, N. B.Y. Tsui, E. K.O. Ng, R. W.K. Chiu, G. M. Joynt, and Y.M. D. Lo Effects of Filtration on Glyceraldehyde-3-Phosphate Dehydrogenase mRNA in the Plasma of Trauma Patients and Healthy Individuals Clin. Chem., January 1, 2004; 50(1): 206 - 208. [Full Text] [PDF]

<