Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: Definition of optimal conditions for specimen collection and clinical application in interferon-treated patients

Davis, Gary L.; Lau, Johnson Y.N.; Urdea, M. S.; Neuwald, Paul; Wilber, Judith C.; Lindsay, Karen L.; Perrillo, Robert P.; Albrecht, Janice K.

doi:10.1002/hep.1840190603

Cited by 176 publications

(63 citation statements)

References 16 publications

(12 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Insulin preRNA and mRNA were quantified using bDNA technology in a 96-microwell format similar to that described for quantification of hepatitis C virus RNA (16). All components, including buffers and DNA reagents were obtained from Chiron.…”

Section: Methodsmentioning

confidence: 99%

Regulation of insulin preRNA splicing by glucose

Wang

Shen

Najafi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Glucose tightly regulates the synthesis and secretion of insulin by ␤ cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single ␤ cell (␤TC3 cells or mouse islets), whereas 1 million non-insulinproducing ␣ cells (␣TC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from ␤ cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up Ϸ2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription.Insulin, the key signal for energy storage, is synthesized and secreted by the ␤ cells of the pancreatic islets of Langerhans in mammals. Insulin synthesis and secretion is tightly regulated by glucose, which serves as a signal of the energy state of the organism. Synthesis of mature insulin is a multistep process and glucose regulates synthesis at several of these steps, including transcription (1), mRNA stabilization (2), translation (3, 4), and processing proinsulin to insulin (5).The level of insulin mRNA available in the cytoplasm for translation to preproinsulin depends on the relative rates of insulin mRNA production and degradation. Glucose regulates both insulin gene transcription and insulin mRNA degradation (1, 2). Transcription rate alone, however, does not determine the production rate of cytoplasmic insulin mRNA. Prior to export from the nucleus, preRNAs are spliced and a 5Ј m 7 GpppN cap and 3Ј adenosines are added. These processes are not independent: splicing accelerates polyadenylylation, and both are important for nuclear export (6-8).Insulin preRNA in most species contains two introns. The sequences of the introns show little evidence of evolutionary conservation, but the positions of the introns are highly conserved (9, 10). The length of intron 2 varies widely among species, but intron 1 is generally small (118 ...

show abstract

Section: Methodsmentioning

confidence: 99%

Regulation of insulin preRNA splicing by glucose

Wang

Shen

Najafi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Phlebotomy centers and laboratories must process samples for viral testing under conditions that do not allow degradation of HCV RNA. 27 Samples should be refrigerated or serum separated from the clot within 2 hours. Quantitative testing should be performed by a method with a wide enough dynamic range to allow for accurate assessment of the pretreatment HCV RNA level.…”

Section: Issues In Using Quantitative Hcv Rna Testing In Clinical Pramentioning

confidence: 99%

Monitoring of viral levels during therapy of hepatitis C

Davis

2002

Hepatology

Self Cite

View full text Add to dashboard Cite

Alpha interferon therapy of chronic hepatitis C is typically accompanied by a biphasic decrease in hepatitis C virus (HCV) RNA levels: an initial rapid decline during the first 24 to 48 hours, and a second more gradual decline during the following weeks. The rate of second-phase decline correlates with ultimate response to interferon treatment. Thus, assessment of early virological response (EVR) may predict outcome. Data from 2 large clinical trials of peginterferon and ribavirin were combined and analyzed to determine the optimal definition of an EVR which, if not achieved, was associated with a low likelihood of a sustained virological response (SVR). A fall in HCV RNA level to undetectable or by at least 2 log 10 units after 12 weeks was found to be the optimal definition of an EVR. Among 965 patients, 778 (80%) achieved an EVR by week 12, including all except 1 patient with genotypes 2 or 3. Among 187 patients without an EVR, only 3 (1.6%) had an SVR. These findings suggest that patients with genotype 1 who do not achieve an EVR should stop treatment after 12 weeks. Use of an early stopping rule reduces treatment costs by at least 16% and avoids the inconvenience and side effects of treatment in the 19% of patients without an EVR who have little chance of a lasting virological response. (HEPATOLOGY 2002; 36:S145-S151.)

show abstract

“…[14][15][16][17][18][19][20] The level of sensitivity achieved by the more recent versions of these technologies has improved qualitative assessment of HCV serum status. Clinically relevant sensitivities of 100 viral genomes per mL of serum are now readily achievable.…”

mentioning

confidence: 99%

Natural fluctuations of hepatitis C viral load in a homogeneous patient population: A prospective study

et al. 2000

View full text Add to dashboard Cite

Hepatitis C virus (HCV) infection is the main cause of parenteral non-A, non-B hepatitis. 1-5 HCV consists of a heterogeneous mix of isolates defined by genotype, each of which is further classified into subtypes. A number of factors have been identified as important in predicting the outcome of disease progression. These include age at infection, viral type/subtype, viral load, quasispecies, and mode of infection. [6][7][8][9][10] Clinical heterogeneity in disease progression may reflect either viral heterogeneity or variations in host response. However, the fluctuations in viral load during the natural history of hepatitis C infection are poorly understood. The purpose of the present study was to determine if viral load remains at a steady state or is in dynamic flux during the course of an HCV infection. To avoid heterogeneity of risk factors and confounding variables in viral type/subtype, we studied a unique cohort of individuals all infected by anti-D/ blood product from a single source of HCV 1b. The sole source of the infectious agent was identified as HCV 1b-contaminated anti-D immunoglobulin. The contaminating HCV 1b was derived from a single donor. [11][12][13] The patients were of similar ethnogeographic background and had an absence of other risk factors for liver disease.Serum HCV RNA can be quantitated by a range of different technologies (reverse transcription-polymerase chain reaction [RT-PCR], RT-PCR coupled to different signal amplification systems such as colorimetric assays, and the branched chain technology). [14][15][16][17][18][19][20] The level of sensitivity achieved by the more recent versions of these technologies has improved qualitative assessment of HCV serum status. Clinically relevant sensitivities of 100 viral genomes per mL of serum are now readily achievable.The work reported here used the Roche Monitor system for amplification of the HCV. The high degree of reproducible quantification of viral load and a uniform linear range of amplification between the different versions of the Roche HCV Monitor assays for HCV of genotype 1b provide a means in which quantifications over several generations of assays are suitable for longitudinal studies. In addition, amplification of HCV genotype 1b by the Monitor system does not suffer from the reported difficulties associated with other genotypes. 21

show abstract

Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: Definition of optimal conditions for specimen collection and clinical application in interferon-treated patients

Cited by 176 publications

References 16 publications

Regulation of insulin preRNA splicing by glucose

Regulation of insulin preRNA splicing by glucose

Monitoring of viral levels during therapy of hepatitis C

Natural fluctuations of hepatitis C viral load in a homogeneous patient population: A prospective study

Contact Info

Product

Resources

About