Developments in Quantitative PCR

Orlando, Claudio; Pinzani, Pamela; Pazzagli, Mario

doi:10.1515/cclm.1998.045

Cited by 296 publications

(163 citation statements)

References 105 publications

(114 reference statements)

Supporting

Mentioning

155

Contrasting

Unclassified

Order By: Relevance

“…The benefits of real time quantitative PCR (25) have been widely described (26,27). These include improved reproducibility, reduced contamination risks and higher assay throughput when compared to traditional quantitative PCR assays that include several post-PCR steps.…”

Section: Discussionmentioning

confidence: 99%

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

Nurmi

Ylikoski

Soukka

et al. 2000

Nucleic Acids Research

View full text Add to dashboard Cite

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5′-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5′→3′ exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10-10 7 initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5′-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.

show abstract

Section: Discussionmentioning

confidence: 99%

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

Nurmi

Ylikoski

Soukka

et al. 2000

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…The probe sequence is FAM-TGG CGA GCC CTC AGA TGC TGC-TAMRA. The ABI PRISM 7700 was cycled at 60°C for 30 min, 95°C for 5 min, 95°C for 15 s (45 cycles), and 60°C for 1 min (37,38). …”

Section: Measurement Of Hiv-1 Replicationmentioning

confidence: 99%

Peroxisome Proliferator-activated Receptor γ Agonists Inhibit HIV-1 Replication in Macrophages by Transcriptional and Post-transcriptional Effects

Hayes¹,

Lane²,

King³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

“…RT-PCR is a time-tested method for studying gene expression and comparing levels of gene expression in different specimens [6]. RT-PCR is especially useful in situations where amounts of RNA are insufficient for Northern blot analysis.…”

Section: Introductionmentioning

confidence: 99%

Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR

Rush

Tan

et al. 2004

Molecular and Biochemical Parasitology

View full text Add to dashboard Cite

Improved understanding of the biology of reproduction in filarial worms may lead to identification of new targets for drugs or vaccines. Real-time RT-PCR is increasingly being adopted for RNA quantification and genetic analysis. Candidate gender-regulated genes were selected from genes identified in prior studies by differential display RT-PCR and by electronic selection of the Brugia malayi expression sequence tag (EST) database for clusters with possible gender-specific expression (four or more transcripts in male cDNA library ESTs but none in female ESTs or vice versa). Expression of candidate genes in male and female worms was compared by real-time reverse transcription PCR with sequence-specific primers. Double stranded DNA product was measured by SYBR Green I fluorescence; melting curves and agarose gel electrophoresis were used to verify the specificity of results. Relative gene expression results were normalized by parallel studies with internal control genes that were shown to be equally expressed in male and female worms (beta actin 2B, histone H3, NADH dehydroxygenase subunit 1) and calculated by the comparative C t method. Nineteen of 31 candidate genes were verified to have reproducible, gender-biased expression with fold differences between 5 and >30,000. These included several well-known genes (for example, genes encoding major sperm protein and a microfilaria sheath protein) and many novel genes. This paper reports the first large scale use of real time RT-PCR to quantitate and study gene expression in a nematode parasite. Our results represent an important step toward improved understanding of the molecular biology of reproduction in filarial nematodes.

show abstract

Developments in Quantitative PCR

Cited by 296 publications

References 105 publications

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

Peroxisome Proliferator-activated Receptor γ Agonists Inhibit HIV-1 Replication in Macrophages by Transcriptional and Post-transcriptional Effects

Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR

Contact Info

Product

Resources

About