Skip to main content

Main menu

  • Home
  • About
    • Clinical Chemistry
    • Editorial Board
    • Most Read
    • Most Cited
    • Alerts
    • CE Credits
  • Articles
    • Current Issue
    • Early Release
    • Future Table of Contents
    • Archive
    • Browse by Subject
  • Info for
    • Authors
    • Reviewers
    • Subscribers
    • Advertisers
    • Permissions & Reprints
  • Resources
    • AACC Learning Lab
    • Clinical Chemistry Trainee Council
    • Clinical Case Studies
    • Clinical Chemistry Guide to Scientific Writing
    • Clinical Chemistry Guide to Manuscript Review
    • Journal Club
    • Podcasts
    • Q&A
    • Translated Content
  • Abstracts
  • Submit
  • Contact
  • Other Publications
    • The Journal of Applied Laboratory Medicine

User menu

  • Subscribe
  • My alerts
  • Log in
  • Log out

Search

  • Advanced search
Clinical Chemistry
  • Other Publications
    • The Journal of Applied Laboratory Medicine
  • Subscribe
  • My alerts
  • Log in
  • Log out
Clinical Chemistry

Advanced Search

  • Home
  • About
    • Clinical Chemistry
    • Editorial Board
    • Most Read
    • Most Cited
    • Alerts
    • CE Credits
  • Articles
    • Current Issue
    • Early Release
    • Future Table of Contents
    • Archive
    • Browse by Subject
  • Info for
    • Authors
    • Reviewers
    • Subscribers
    • Advertisers
    • Permissions & Reprints
  • Resources
    • AACC Learning Lab
    • Clinical Chemistry Trainee Council
    • Clinical Case Studies
    • Clinical Chemistry Guide to Scientific Writing
    • Clinical Chemistry Guide to Manuscript Review
    • Journal Club
    • Podcasts
    • Q&A
    • Translated Content
  • Abstracts
  • Submit
  • Contact
Research ArticleArticle

Presence of Filterable and Nonfilterable mRNA in the Plasma of Cancer Patients and Healthy Individuals

Enders K.O. Ng, Nancy B.Y. Tsui, Nicole Y.L. Lam, Rossa W.K. Chiu, Simon C.H. Yu, S.C. Cesar Wong, Elena S.F. Lo, Timothy H. Rainer, Philip J. Johnson, Y.M. Dennis Lo
Published August 2002
Enders K.O. Ng
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nancy B.Y. Tsui
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nicole Y.L. Lam
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rossa W.K. Chiu
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Simon C.H. Yu
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
S.C. Cesar Wong
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Elena S.F. Lo
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Timothy H. Rainer
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Philip J. Johnson
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Y.M. Dennis Lo
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Background: As RNA is labile, we investigated whether circulating RNA in human plasma may be present in a particle-associated form.

Methods: Blood was collected from 27 healthy individuals and 16 hepatocellular carcinoma (HCC) patients. The plasma from each individual was processed by two means: filtration through filters with different pore sizes (from 5 μm to 0.22 μm) and ultracentrifugation. We assessed plasma RNA content by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts and plasma DNA by a real-time quantitative PCR assay for the β-globin gene.

Results: The plasma GAPDH mRNA concentrations in the healthy individuals were significantly different in every pair of these filter sizes (P <0.05 for each pair). Overall, the plasma GAPDH mRNA concentration was higher by a median of 15-fold (interquartile range, 10- to 24-fold) in the paired unfiltered sample than in the sample filtered through a 0.22 μm filter. In contrast, no significant difference was seen in β-globin DNA concentrations among different pore-size-filtered plasma samples (P = 0.455). Similarly, a significant difference was observed for RNA, but not DNA, between unfiltered plasma and ultracentrifuged plasma (P <0.05). No significant difference in GAPDH mRNA concentrations was seen between the 0.22-μm-filtered plasma samples and the ultracentrifuged plasma samples (P >0.05). In HCC patients, filtration with a 0.22 μm filter produced a median 9.3-fold (interquartile range, 6.9- to 311-fold) reduction in GAPDH mRNA concentration in plasma. Plasma GAPDH mRNA concentrations in HCC patients were significantly higher than those in healthy individuals, both with or without filtration (P <0.0 5 for filtered plasma samples; P <0.005 for unfiltered plasma samples).

Conclusions: A substantial proportion of plasma mRNA species is particle-associated. In HCC patients, both circulating particle- and non-particle-associated plasma RNA are increased.

Much interest has been focused on the diagnostic applications and biology of circulating nucleic acids (1). A recent development in this field is the discovery of tumor-derived RNA in the plasma/serum of cancer patients (2)(3)(4)(5)(6). Tumor-associated RNA targets that have been detected include mRNA of the tyrosinase gene (2), telomerase components (4)(6), and viral RNA (3). The lability of RNA and the existence of ribonuclease in the plasma (7) make it surprising that circulating RNA should be detectable at all. Hasselmann et al. (8) recently made the interesting observation that in an in vitro model, mRNA within apoptotic bodies released by a melanoma cell line was protected from degradation when incubated in human serum. However, a conclusive demonstration that circulating RNA in human plasma is particle-associated is currently lacking. Such incomplete understanding of the fundamental biology of circulating RNA is compounded by the fact that all except one (6) of the recent publications on circulating RNA are qualitative studies. In this report, we address both of these issues, using a quantitative real-time reverse transcription-PCR (RT-PCR)1 to measure circulating RNA in cancer patients and healthy individuals. In particular, we investigated the possibility that circulating RNA may be present in a particle-associated form by analyzing plasma samples that have been processed by filtration and ultracentrifugation.

Materials and Methods

patients

Sixteen hepatocellular carcinoma (HCC) patients undergoing investigation at the Department of Diagnostic Radiology and Imaging at the Prince of Wales Hospital were recruited with informed consent. The healthy control group consisted of 27 individuals. The study was approved by the Ethics Committee of The Chinese University of Hong Kong.

processing of blood samples

Our current protocol, based on our previous report that blood processing by different protocols would affect the results of plasma DNA quantification (9), involves initial centrifugation of blood samples at 1600g followed by a second centrifugation at 16 000g. This protocol has been shown to minimize the contribution of DNA derived from residual cells in plasma (9). Peripheral blood was collected from each participant into EDTA tubes. Blood samples were centrifuged at 1600g for 10 min at 4 °C, and plasma was carefully transferred into new tubes, followed by further centrifugation at 16 000g for 10 min at 4 °C.

Plasma samples from 17 healthy individuals were used in the filtration experiments. Each plasma sample was divided into four aliquots: three were individually passed through filters (Millex-GV; Millipore) with pore sizes of 0.22, 0.45, and 5 μm. The remaining aliquot was not subjected to filtration.

The plasma samples from 10 additional healthy individuals were used in the filtration and ultracentrifugation experiments. Each sample was divided into three aliquots: one-third of the plasma was filtered with a 0.22 μm filter, and one-third was subjected to ultracentrifugation (using a Beckman OptimaTM TLX Ultracentrifuge) at 99 960g for 120 min at 4 °C. The remaining aliquot was not filtered or subjected to ultracentrifugation.

The plasma sample from each HCC patient was divided into two aliquots: one-half was filtered with a 0.22 μm filter, and the remaining aliquot remained unfiltered.

rna extraction from plasma samples

RNA was extracted from 600 μL of plasma with an RNeasy Mini Kit (Qiagen). Briefly, 600 μL of plasma was mixed with 1.2 mL of Trizol LS reagent (Invitrogen) and 0.2 mL of chloroform. The mixture was centrifuged at 11 900g for 15 min at 4 °C, and the aqueous layer was transferred into new tubes. One volume of 700 mL/L ethanol was added to one volume of the aqueous layer. The mixture was then applied to the RNeasy minicolumn and was processed according to the manufacturer’s recommendations. Total RNA was eluted with 15 μL of RNase-free water followed by DNase I (Invitrogen) treatment. RNA was stored at −80 °C until further processing.

dna extraction from plasma samples

Plasma DNA was extracted with a QIAamp Blood Kit (Qiagen), using the “blood and body fluid protocol” as recommended by the manufacturer (10). A final elution volume of 25 μL was used.

real-time quantitative rt-pcr

One-step real-time quantitative RT-PCR was used for the measurement of mRNA concentration in plasma (11). In this method, the rTth DNA polymerase functioned both as a reverse transcriptase and a DNA polymerase. A real-time quantitative RT-PCR method was used for transcript quantification of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The amplification primers were GAPDHF (5′-GAAGGTGAAGGTCGGAGT-3′) and GAPDHR (5′-GAAGATGGTGATGGGATTTC-3′), and the dual-labeled fluorescent probe was GAPDHP [5′-(FAM)CAAGCTTCCCGTTCTCAGCC(TAMRA)-3′], where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine.

RT-PCR was set up in a reaction volume of 50 μL using components supplied in an EZ rTth RNA PCR reagent set and TaqMan® GAPDH Control Reagents (Human; PE Applied Biosystems). Each reaction contained 10 μL of 5× EZ buffer; 200 nM each of the primers; 100 nM fluorescent probe; 3 mM Mn(OAc)2; 300 μM each of dATP, dCTP, and dGTP; 600 μM dUTP; 5 U of rTth polymerase; and 0.5 U of uracil N-glycosylase. For amplification, we used 3 μL of extracted RNA. Amplification data were collected and analyzed with an ABI Prism 7700 Sequence Detector (PE Applied Biosystems). Each sample was analyzed in duplicate, and multiple negative water blanks were included in every analysis. A calibration curve for GAPDH quantification was prepared by subjecting serial dilutions of human control RNA (PE Applied Biosystems), with RNA concentrations ranging from 15 to 0.23 pg, to the RT-PCR assay. The manufacturer estimated that 1 pg of this control RNA contained ∼100 copies of GAPDH transcript.

The thermal profile used for the GAPDH method was as follows: before reverse transcription, the reaction was initiated at 50 °C for 2 min in the presence of uracil N-glycosylase, followed by a reverse transcription step at 60 °C for 30 min. After a 5-min denaturation at 95 °C, PCR was carried out for 40 cycles using a denaturation step of 94 °C for 20 s and an annealing/extension step of 60 °C for 1 min.

real-time quantitative dna pcr

Plasma DNA was quantified by a real-time quantitative PCR for the β-globin gene as described previously (10). For each reaction, 5 μL of the extracted plasma DNA was used and analyzed in duplicate. Thermal cycling conditions for the β-globin PCR were as described previously (10). The precision of this method has been reported previously, with a CV for the threshold cycle of 1.1% (10).

statistical analysis

Statistical analysis was performed using SigmaStat 2.03 software (SPSS).

Results

validation of real-time quantitative rt-pcr

To assess the linearity of the assay, we analyzed serial dilutions of the control RNA, using the GAPDH RT-PCR method. The calibration curve showed a correlation coefficient of 0.995. To assess the precision of both the RNA extraction and the RT-PCR steps, we prepared 10 replicate extracts from a plasma sample obtained from a healthy individual; these extracted RNA samples were then subjected to real-time quantitative RT-PCR analysis. The CV for the threshold cycle values of these replicate analyses was 1.7%. The lower limit of detection for this RT-PCR method was ∼23 copies, the number of GAPDH transcripts present in 0.23 pg of the control RNA.

particle-associated nature of circulating rna in the plasma of healthy individuals

Plasma samples from 17 healthy individuals were analyzed for GAPDH mRNA and β-globin DNA concentrations. Aliquots from each sample were individually passed through filters with pore sizes of 5, 0.45, and 0.22 μm before quantitative analysis. The data in Fig. 1A⇓ show that filtration had a clearly observable effect on GAPDH mRNA concentrations in plasma samples (Friedman test, P <0.001). Pairwise analysis further indicated that a statistically significant difference was detected in every pair of these filter sizes (Student-Newman-Keuls test, P <0.05 for each pair). Overall, the plasma GAPDH mRNA concentration decreased by a median of 15-fold (interquartile range, 10- to 24-fold) in comparisons of paired samples from the unfiltered samples and samples filtered through the 0.22 μm filters. In contrast to these results, there was no statistically significant difference in β-globin DNA concentrations among plasma samples filtered through different-sized pores (Friedman test, P = 0.455; Fig. 1B⇓ ). These results therefore indicate that a significant proportion of GAPDH mRNA in plasma is particle-associated. On the other hand, most of the circulating β-globin DNA is non-particle-associated.

Figure 1.
  • Download figure
  • Open in new tab
Figure 1.

Plasma mRNA (A) and DNA (B) concentrations in healthy individuals after filtration through different pore sizes.

(A), plasma GAPDH mRNA concentrations (ng/L), as determined by real-time quantitative RT-PCR (y axis), plotted against filter pore size (x axis). (B), plasma β-globin DNA concentrations (genome-equivalents/mL), as determined by real-time quantitative PCR (y axis), plotted against filter pore size (x axis). The lines inside the boxes denote medians. The boxes mark the interval between the 25th and 75th percentiles. The whiskers denote the interval between the 10th and 90th percentiles. • indicate data points outside the 10th and 90th percentiles.

To investigate whether any mRNA in plasma existed in a non-particle-associated form, we performed a second series of experiments using ultracentrifugation. Plasma samples were obtained from another group of 10 healthy individuals. Aliquots from each sample were subjected to either a 0.22 μm filtration step or ultracentrifugation at 99 960g. The data in Fig. 2⇓ show a statistically significant difference among GAPDH mRNA concentrations in unfiltered plasma, plasma filtered through a 0.22 μm filter, and ultracentrifuged plasma (Friedman test, P <0.001). Pairwise comparisons indicated a significant difference between unfiltered plasma and plasma filtered through 0.22 μm filters (Student-Newman-Keuls test, P <0.05), and between unfiltered plasma and ultracentrifuged plasma (Student-Newman-Keuls test, P <0.05). However, there was no significant difference in GAPDH mRNA concentrations between the plasma samples filtered through 0.22 μm filters and the ultracentrifuged plasma samples (Student-Newman-Keuls test, P >0.05; Fig. 2A⇓ ). The median fractional concentration of GAPDH mRNA in the supernatants of the ultracentrifuged plasma samples was 7.4% (interquartile range, 4.3–13%) that of the unfiltered samples. On the other hand, the differences in β-globin DNA concentrations among the samples subjected to different treatments did not reach statistical significance (Friedman test, P = 0.122).

Figure 2.
  • Download figure
  • Open in new tab
Figure 2.

Plasma mRNA (A) and DNA (B) concentrations in healthy individuals after filtration and ultracentrifugation treatments.

(A), plasma GAPDH mRNA concentrations (ng/L), as determined by real-time quantitative RT-PCR (y axis), plotted against the processing categories (x axis). (B), plasma β-globin DNA concentrations (genome-equivalents/mL), as determined by real-time quantitative PCR (y axis), plotted against the processing categories (x axis). Untreated, no treatment; Filtered, filtered through 0.22 μm filter; Ultracentrifuged, ultracentrifugation at 99 960g. The lines inside the boxes denote the medians. The boxes mark the interval between the 25th and 75th percentiles. The whiskers denote the interval between the 10th and 90th percentiles. • indicate data points outside the 10th and 90th percentiles.

quantitative analysis of GAPDH mRNA in the plasma of hcc patients

To determine whether similar results could be obtained for cancer patients, we analyzed plasma samples from 16 HCC patients. As shown in Fig. 3⇓ , filterable GAPDH mRNA was present in the plasma of HCC patients. Indeed, filtration with a 0.22 μm filter produced a median 9.3-fold (interquartile range, 6.9- to 311-fold) decrease in GAPDH mRNA concentration in plasma. The data in Fig. 3A⇓ also indicate that plasma GAPDH mRNA concentrations in HCC patients were significantly higher than those in healthy individuals, both with or without filtration (Mann-Whitney rank-sum test, P <0.05 for filtered plasma samples and P <0.005 for unfiltered plasma samples). In 7 of the 16 HCC cases, sufficient plasma samples had been collected for an additional experiment involving plasma β-globin DNA quantification. No statistically significant difference was observed for plasma β-globin DNA concentrations between HCC patients and healthy individuals, both with or without filtration (Mann-Whitney rank-sum test, P = 0.525 for filtered plasma samples and P = 0.418 for unfiltered plasma samples; Fig. 3B⇓ ).

Figure 3.
  • Download figure
  • Open in new tab
Figure 3.

Comparison of plasma mRNA and DNA concentrations in healthy individuals (□) and HCC patients (▧) with or without filtration through 0.22 μm filters.

The processing categories are shown on the x axis. (A), plasma GAPDH mRNA concentrations (ng/L), as determined by real-time quantitative RT-PCR, are plotted on the y axis (common logarithmic scale). (B), plasma β-globin DNA concentrations (genome-equivalents/mL), as determined by real-time quantitative PCR, are plotted on the y axis (common logarithmic scale). The lines inside the boxes denote the medians. The boxes mark the interval between the 25th and 75th percentiles. The whiskers denote the interval between the 10th and 90th percentiles. • indicate data points outside the 10th and 90th percentiles.

Discussion

The discovery of circulating RNA in the plasma/serum of healthy individuals and cancer patients has opened up new possibilities for noninvasive cancer detection and monitoring (2)(3)(4)(5)(6)(12)(13). Currently, little is known about the characteristics and biological origin of circulating RNA. Cell death, including apoptosis, has been postulated to be one of the mechanisms responsible for the release of nucleic acids into the plasma (14). Because cells undergoing apoptosis dispose nucleic acids into apoptotic bodies (15), it is possible that at least part of the circulating RNA is particle-bound and, thus, potentially protected from nuclease degradation (8). This hypothesis is particularly likely in view of the recent demonstration of apoptotic cells in human plasma (16).

In this study, we investigated the particle-associated nature of circulating RNA by subjecting plasma samples to filtration through filters with different pore sizes. Our data clearly indicate that filterable GAPDH mRNA species are present in human plasma. Thus, the median concentration of plasma GAPDH mRNA was decreased 1.4-fold in samples passed through a 5 μm filter. An additional 2.4-fold reduction was observed when the data for the samples filtered through the 5 μm filters were compared with the results obtained for samples filtered through a 0.45 μm filter. The greatest reduction in GAPDH signal was observed after filtration through a 0.22 μm filter; the median concentration after filtration through the 0.22 μm filters was 5.1-fold lower than in samples filtered through 0.45 μm filters.

The existence of particle-associated circulating mRNA species raises the question as to whether all circulating mRNA species exist in this fashion. This question is relevant to the GAPDH mRNA signal, which was still detectable after filtration through a 0.22 μm filter (Fig. 1A⇑ ). To address this question, we performed a series of experiments in which we measured the concentrations of GAPDH mRNA in plasma that had been subjected to ultracentrifugation. In a previous study, Yamamoto et al. (17) used a centrifugal force of 70 000g to pellet viral particles. The higher centrifugal force (99 960g) used in our study would therefore be expected to pellet virtually all particulate matter. It is interesting to note, therefore, that even after this ultracentrifugation step, a small amount of GAPDH mRNA was still detectable in the supernatant. This signal represented a median of 7.4% of the original GAPDH mRNA concentration present in the nonultracentrifuged plasma sample and was likely to represent non-particle-associated circulating mRNA species. In the present study, we did not determine whether these non-particle-associated mRNA species might be protected from degradation by complexing with protein or lipid moieties.

With particular relevance to cancer detection and monitoring, we also demonstrated that the phenomenon of particle-associated plasma mRNA species can also be observed in cancer patients. Thus, filtration with a 0.22 μm filter produced a marked decrease in plasma GAPDH mRNA concentrations in HCC patients (Fig. 3A⇑ ). Interestingly, we also found that with or without filtration, GAPDH mRNA concentrations in the plasma of HCC patients were significantly higher than those in healthy individuals (Fig. 3A⇑ ). These results suggest that the plasma of HCC patients contains increased concentrations of both particle-associated and non-particle-associated mRNA species. It is tempting to speculate that such mRNA species are released by the tumor cells, although confirmation of this hypothesis requires future experimentation. With regard to the particle-associated mRNA species, one possibility is that apoptosis of neoplastic cells in cancer patients might release apoptotic bodies packaged with RNA into plasma.

Our demonstration of particle-associated mRNA in plasma indicates that this component should be taken into account in future studies involving circulating mRNA. Apart from HCC, it is also important that similar experiments be carried out for other cancer types. It would also be of interest to investigate the relationship between the relative amount of particle-associated and non-particle-associated mRNA species and clinical variables such as tumor stage and treatment response. The phenomenon of particle-associated plasma RNA should also be studied in other scenarios in which circulating mRNA has been detected, such as fetal RNA in maternal plasma (18).

Future research is needed to elucidate the nature of such particle-associated mRNA in plasma. Techniques such as flow cytometry and conventional and electron microscopy might provide further physical information on such particles. Another important issue concerns the cellular origin of such particle-associated mRNA. In this regard, our recent demonstration in a bone marrow transplantation model that plasma DNA is predominantly hematopoietic in origin (19) would suggest that one should perhaps start by studying mRNA species of hematopoietic origin.

In addition to our GAPDH mRNA findings, we also determined that with or without filtration, there is no significant difference in plasma β-globin DNA concentrations between cancer patients and healthy individuals. In contrast to mRNA, most β-globin DNA in plasma is nonfilterable, which is consistent with our previous study that the double centrifugation protocol used in this study (1600g followed by 16 000g) would produce no significant difference in β-globin DNA concentrations between filtered and unfiltered plasma samples (9).

There are potentially many possible explanations for the apparent difference in the particle- and non-particle-associated nature of circulating RNA and DNA. One possible hypothesis is that both DNA and RNA are initially released into the plasma as both particle- and non-particle-associated forms. We would further hypothesize that the non-particle-associated forms are present in much higher concentrations than the particle-associated forms. By extension of the work by Hasselmann et al. (8), we would propose that the particle-associated forms of DNA and RNA are protected from degradation. If, as we hypothesize, the non-particle-associated form of DNA is released in substantially higher concentrations than the particle-associated forms, any quantitative removal of the latter by filtration will be “masked” by the overwhelming concentration of the former. However, because of the lability of RNA, the situation for this type of nucleic acid would be different. Thus, we would suggest that, because of its labile nature, most of the non-particle-associated RNA is degraded after release. The RNA species that are left would therefore be predominantly the relatively protected particle-associated forms. The predominance of such particle-associated RNA would thus explain the results of the filtration and ultracentrifugation experiments described in this report. Obtaining conclusive evidence to support this hypothetical model would require further experimentation.

To our knowledge, this study represents the second study of plasma RNA using quantitative techniques. The only previous quantitative study was based on the measurement of telomerase reverse transcriptase mRNA in the plasma of cancer patients (6). It is important to note that in the previous study, the results were expressed as a ratio of telomerase reverse transcriptase mRNA to GAPDH mRNA. Our demonstration of increased particle-associated and non-particle-associated GAPDH mRNA concentrations in the plasma of HCC patients indicates that caution should be exercised when expressing or interpreting plasma RNA data as a ratio. Indeed, we believe that until the precise elucidation of the distribution of particle-associated and non-particle-associated mRNA species of different genes in the plasma of cancer patients is accomplished, it would be prudent to express quantitative mRNA measurements in plasma as an absolute concentration.

Acknowledgments

The work is supported by the Innovation and Technology Fund (AF/90/99). We thank Lisa Chan, Allen Chan, Katherine Chow, and Blenda Wong for help and advice during this study.

Footnotes

  • ↵1 Nonstandard abbreviations: RT-PCR, reverse transcription-PCR; HCC, hepatocellular carcinoma; and GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

  • © 2002 The American Association for Clinical Chemistry

References

  1. ↵
    Lo YMD. Circulating nucleic acids in plasma and serum: an overview. Ann N Y Acad Sci 2001;945:1-7.
    OpenUrlCrossRefPubMed Order article via Infotrieve
  2. ↵
    Kopreski MS, Benko FA, Kwak LW, Gocke CD. Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin Cancer Res 1999;5:1961-1965.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Lo KW, Lo YMD, Leung SF, Tsang YS, Chan LY, Johnson PJ, et al. Analysis of cell-free Epstein-Barr virus associated RNA in the plasma of patients with nasopharyngeal carcinoma. Clin Chem 1999;45:1292-1294.
    OpenUrlFREE Full Text
  4. ↵
    Chen XQ, Bonnefoi H, Pelte MF, Lyautey J, Lederrey C, Movarekhi S, et al. Telomerase RNA as a detection marker in the serum of breast cancer patients. Clin Cancer Res 2000;6:3823-3826.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Silva JM, Dominguez G, Silva J, Garcia JM, Sanchez A, Rodriguez O, et al. Detection of epithelial messenger RNA in the plasma of breast cancer patients is associated with poor prognosis tumor characteristics. Clin Cancer Res 2001;7:2821-2825.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Dasi F, Lledo S, Garcia-Granero E, Ripoll R, Marugan M, Tormo M, et al. Real-time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA: a simple blood test to monitor disease in cancer patients. Lab Invest 2001;81:767-769.
    OpenUrlPubMed Order article via Infotrieve
  7. ↵
    Reddi KK, Holland JF. Elevated serum ribonuclease in patients with pancreatic cancer. Proc Natl Acad Sci U S A 1976;73:2308-2310.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Hasselmann DO, Rappl G, Tilgen W, Reinhold U. Extracellular tyrosinase mRNA within apoptotic bodies is protected from degradation in human serum. Clin Chem 2001;47:1488-1489.
    OpenUrlFREE Full Text
  9. ↵
    Chiu RWK, Poon LL, Lau TK, Leung TN, Wong EM, Lo YMD. Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 2001;47:1607-1613.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Lo YMD, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768-775.
    OpenUrlCrossRefPubMed Order article via Infotrieve
  11. ↵
    Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 1991;88:7276-7280.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Rieber M, Bacalao J. An “external” RNA removable from mammalian cells by mild proteolysis. Proc Natl Acad Sci U S A 1974;71:4960-4964.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Guin LW, Griswold KE, Patton S, Kamm RC, Smith AG. Electrophoretic characterization of plasma RNA. Biochem Med 1975;13:224-230.
    OpenUrlCrossRefPubMed Order article via Infotrieve
  14. ↵
    Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 2001;61:1659-1665.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Halicka HD, Bedner E, Darzynkiewicz Z. Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis. Exp Cell Res 2000;260:248-256.
    OpenUrlCrossRefPubMed Order article via Infotrieve
  16. ↵
    van Wijk IJ, De Hoon AC, Jurhawan R, Tjoa ML, Griffioen S, Mulders MAM, et al. Detection of apoptotic fetal cells in plasma of pregnant women. Clin Chem 2000;46:729-731.
    OpenUrlFREE Full Text
  17. ↵
    Yamamoto M, Kimura H, Hironaka T, Hirai K, Hasegawa S, Kuzushima K, et al. Detection and quantification of virus DNA in plasma of patients with Epstein-Barr virus-associated diseases. J Clin Microbiol 1995;33:1765-1768.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Poon LL, Leung TN, Lau TK, Lo YMD. Presence of fetal RNA in maternal plasma. Clin Chem 2000;46:1832-1834.
    OpenUrlFREE Full Text
  19. ↵
    Lui YY, Chik KW, Chiu RW, Ho CY, Lam CW, Lo YMD. Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin Chem 2002;48:421-427.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top

In this issue

Clinical Chemistry: 48 (8)
Vol. 48, Issue 8
August 2002
  • Table of Contents
  • Index by author
Print
Share
Presence of Filterable and Nonfilterable mRNA in the Plasma of Cancer Patients and Healthy Individuals
Enders K.O. Ng, Nancy B.Y. Tsui, Nicole Y.L. Lam, Rossa W.K. Chiu, Simon C.H. Yu, S.C. Cesar Wong, Elena S.F. Lo, Timothy H. Rainer, Philip J. Johnson, Y.M. Dennis Lo
Clinical Chemistry Aug 2002, 48 (8) 1212-1217;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Article Alerts
Sign In to Email Alerts with your Email Address
Citation Tools
Presence of Filterable and Nonfilterable mRNA in the Plasma of Cancer Patients and Healthy Individuals
Enders K.O. Ng, Nancy B.Y. Tsui, Nicole Y.L. Lam, Rossa W.K. Chiu, Simon C.H. Yu, S.C. Cesar Wong, Elena S.F. Lo, Timothy H. Rainer, Philip J. Johnson, Y.M. Dennis Lo
Clinical Chemistry Aug 2002, 48 (8) 1212-1217;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero

  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Comparative Analysis of Biochemical Biases by Ligation- and Template-Switch-Based Small RNA Library Preparation Protocols
  • A Novel Online Calculator Based on Serum Biomarkers to Detect Hepatocellular Carcinoma among Patients with Hepatitis B
  • Clinical Utility of Procalcitonin in the Diagnosis of Pneumonia
Show more Articles

Similar Articles

Subjects

  • SUBJECT AREAS
    • Molecular Diagnostics and Genetics
    • Cancer Diagnostics (since 2002)

Options

  • Home
  • About
  • Articles
  • Information for Authors
  • Resources
  • Abstracts
  • Submit
  • Contact
  • RSS

Other Publications

  • The Journal of Applied Laboratory Medicine
Footer logo

© 2019 American Association for Clinical Chemistry

Powered by HighWire