Quantitation of Target Molecules from Polymerase Chain Reaction-Based Limiting Dilution Assays

Rodrigo, Allen G.; Goracke, Paul C.; Rowhanian, Kiarash; Mullins, James I.

doi:10.1089/aid.1997.13.737

Cited by 129 publications

(103 citation statements)

References 12 publications

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“…Relative copy numbers of each transcript were determined as described previously using the computer program 'QUALITY'. 26 The general assay variability for DC-SIGN and DC-SIGNR, full-length and isoform, mRNA copy number determination was estimated by repeating the complete limiting dilution RT-PCR five times in two tissue donors. Fold-differences between the minimum and maximum copy numbers across each set of replicates ranged from 2.34 to 6.15 (mean 4.1).…”

Section: Genotyping Of Dc-sign and Dc-signr Repeat Region Polymorphismsmentioning

confidence: 99%

Most DC-SIGNR transcripts at mucosal HIV transmission sites are alternatively spliced isoforms

et al. 2005

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The repeat region of DC-SIGNR (CD209L) is polymorphic on the genomic level, and, in a separate study, we observed a correlation between the DC-SIGNR genotype and HIV-1 susceptibility during sexual contact. However, previous investigations using immunohistochemistry failed to detect membrane-bound DC-SIGNR on cells in the genital and rectal mucosa. We therefore explored the presence of DC-SIGNR in these compartments with a more sensitive limiting dilution RT-PCR, which also allowed for quantification of alternatively spliced mRNA isoforms. DC-SIGN (CD209) and DC-SIGNR mRNA transcript isoforms were found in all 12 vaginal and two rectal biopsies obtained from 14 healthy individuals. For DC-SIGNR, we detected significantly more isoform than full-length transcripts (mean copy numbers/lg RNA: 602 vs 26; P ¼ 0.0009). Four mucosal samples lacked full-length DC-SIGNR transcripts entirely. Cloning and sequencing of DC-SIGNR mRNA in three additional individuals revealed a diverse repertoire of DC-SIGNR isoforms, many of which encoded for proteins predicted to be soluble and secreted. Indeed, in one vaginal sample, we detected only soluble isoforms. In conjunction with our prior observation that the DC-SIGNR genotype has an effect on HIV-1 transmission in vivo, these findings emphasize that DC-SIGNR, in addition to DC-SIGN, should be considered as a cofactor in sexual HIV-1 transmission. Soluble isoforms, in particular, may modulate the efficiency of viral transmission and dissemination.

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Section: Genotyping Of Dc-sign and Dc-signr Repeat Region Polymorphismsmentioning

confidence: 99%

Most DC-SIGNR transcripts at mucosal HIV transmission sites are alternatively spliced isoforms

et al. 2005

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“…The third method, denoted PCRbased limited dilution assay, is also aimed at providing individual sequences but avoiding the cloning steps. This is achieved through limiting dilutions prior to PCR amplification (Rodrigo et al, 1997;Taswell, 1981), thus assuring that only a single molecule acts as template for the reaction. Later, these PCR products are sequenced directly.…”

Section: Introductionmentioning

confidence: 99%

Sampling and repeatability in the evaluation of hepatitis C virus genetic variability

Torres-Puente

Bracho

Jiménez

et al. 2003

Journal of General Virology

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Among the experimental techniques available to study the genetic variability of RNA virus populations, the most informative involve reverse transcription (RT), amplification, cloning and sequencing. The effects of several aspects of these techniques on the estimation of genetic variability in a virus population were analysed. Hepatitis C virus populations from four patients were examined. For each patient, ten series of data derived from independent PCR amplifications of a single RT reaction were obtained. The sample size of each data set was 10 sequences (in nine series) and 100 sequences (in one series). An additional data set derived from an independent RT reaction (about 10 sequences) performed on RNA extracted from the same serum sample was also analysed. The availability of data sets of different sample sizes allowed the effect of sample size on the amount and nature of the genetic variability recovered to be examined. The repeatability of the data obtained in different amplification experiments as well as from different RT reactions was also determined, together with the best strategy to obtain a given number of sequences by comparing the set of 100 sequences obtained from a single amplification with those obtained by pooling the nine sets of 10 sequences. In all cases, these results confirm the high repeatability of the conclusions and parameters derived from the sets of 10 sequences. These results validate the use of relatively small sample sets for the evaluation of genetic variability and for the estimation of phylogenetic relationships of RNA viruses in population and epidemiological studies.

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“…Proviral DNA or cDNA was amplified as a 660-bp fragment in the C2-V5 region of env by PCR using primers C/H and D/F (14). Determination of proviral copy numbers was done using a nested PCR-based limiting dilution assay (PLDA), employing a two-pass strategy and the QUALITY statistical computer program (40,53). HIV-1 virion RNA in EDTA-treated plasma was measured with the Amplicor HIV-1 Monitor test kit (Roche Diagnostics Corp., Indianapolis, Ind.)…”

Section: Methodsmentioning

confidence: 99%

Differential Selection of Specific Human Immunodeficiency Virus Type 1/JC499 Variants after Mucosal and Parenteral Inoculation of Chimpanzees

Wei

Fultz

2002

J Virol

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Regardless of the route of transmission, it is generally accepted that the human immunodeficiency virus type 1 (HIV-1) quasispecies transmitted from an infected individual to an uninfected individual is genetically homogeneous. This finding and the observation that HIV-1 genotypes in recipients are minor variants in the donors suggest strongly that selection for specific variants occurs. However, most analyses have been limited to the V3 region of env. In addition, the exact time at which most new infections occurred was not known, making it almost impossible to analyze virus populations present in donor-recipient pairs at the time of HIV-1 transmission. To circumvent this problem, three chimpanzees were inoculated with a genetically defined stock of cell-free HIV-1/JC499 by one of three routes: intravenously or via the cervical or penile mucosa. PCR products of the C2-to-V5 region of env were amplified from both proviral DNA and virion RNA in blood samples collected soon after infection and were screened by heteroduplex analysis (HDA). Those PCR products with distinct HDA banding patterns were cloned and sequenced. In all three animals, transmitted variants encoded one of two V3-loop populations identified in the inoculum, indicating relative homogeneity in this region. However, different virus populations, defined by combinations of specific V4 and V5 sequences, were found when variants in the animal inoculated intravenously (at least 13 V4-plus-V5 combinations) were compared with those in the two animals inoculated by the mucosal routes (limited to only four V4-plus-V5 combinations). The only V4-plus-V5 population in variants found in all three chimpanzees was the major population in the inoculum, which contained viruses with more than 30 different V4-plus-V5 combinations. That the majority of the V4-plus-V5 genotypes in variants transmitted to all three animals were minor populations in the inoculum indicated that selective transmission defined by the V4-plus-V5 regions had occurred but that distinct populations were transmitted by parenteral versus mucosal routes. These results indicate that the putative homogeneity of HIV-1 variants in a newly infected individual might be an artifact of the region of the env gene evaluated and that regions other than V3 might play a major role in selective transmission.

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Quantitation of Target Molecules from Polymerase Chain Reaction-Based Limiting Dilution Assays

Cited by 129 publications

References 12 publications

Most DC-SIGNR transcripts at mucosal HIV transmission sites are alternatively spliced isoforms

Most DC-SIGNR transcripts at mucosal HIV transmission sites are alternatively spliced isoforms

Sampling and repeatability in the evaluation of hepatitis C virus genetic variability

Differential Selection of Specific Human Immunodeficiency Virus Type 1/JC499 Variants after Mucosal and Parenteral Inoculation of Chimpanzees

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