The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

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Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

III. Foldamer Research 3910 A. Overview 3910 B. Motivation 3910 C. Methods 3910 D. General Scope 3912 IV. Peptidomimetic Foldamers 3912 A. The R-Peptide Family 3913 1. Peptoids 3913 2. N,N-Linked Oligoureas 3914 3. Oligopyrrolinones 3915 4. Oxazolidin-2-ones 3916 5. Azatides and Azapeptides 3916 B. The â-Peptide Family 3917 1. â-Peptide Foldamers 3917 2. R-Aminoxy Acids 3937 3. Sulfur-Containing â-Peptide Analogues 3937 4. Hydrazino Peptides 3938 C. The γ-Peptide Family 3938 1. γ-Peptide Foldamers 3938 2. Other Members of the γ-Peptide Family 3941 D. The δ-Peptide Family 3941 1. Alkene-Based δ-Amino Acids 3941 2. Carbopeptoids 3941 V. Single-Stranded Abiotic Foldamers 3944 A. Overview 3944 B. Backbones Utilizing Bipyridine Segments 3944 1. Pyridine−Pyrimidines 3944 2. Pyridine−Pyrimidines with Hydrazal Linkers 3945

A field guide to foldamers.

The long wavelength (far-red to NIR) analyte-responsive fluorescent probes are advantageous for in vivo bioimaging because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence by biomolecules in the living systems. Thus, great interest in the development of new long wavelength analyte-responsive fluorescent probes has emerged in recent years. This review highlights the advances in the development of far-red to NIR fluorescent probes since 2000, and the probes are classified according to their organic dye platforms into various categories, including cyanines, rhodamine analogues, BODIPYs, squaraines, and other types (240 references).

Far-red to near infrared analyte-responsive fluorescent probes based on organic fluorophore platforms for fluorescence imaging

Design strategies for water-soluble small molecular chromogenic and fluorogenic probes.

Near-infrared (NIR) fluorescent dyes have emerged as promising modalities for monitoring the levels of various biologically relevant species in cells and organisms. The use of NIR probes enables deep photon penetration in tissue, minimizes photo-damage to biological samples, and produces low background auto-fluorescence from biomolecules present in living systems. The number of new analyte-responsive NIR fluorescent probes has increased substantially in recent years as a consequence of intense research efforts. In this tutorial review, we highlight recent advances (2010–2013) made in the development and applications of NIR fluorescent probes. The review focuses on NIR fluorescent probes that have been devised to sense various biologically important species, including ROS/RNS, metal ions, anions, enzymes and other related species, as well as intracellular pH changes. The basic principles involved in the design of functional NIR fluorescent probes and suggestions about how to expand applications of NIR imaging agents are also described.

Recent progress in the development of near-infrared fluorescent probes for bioimaging applications

The effects of anthryl derivatives and 9-aminoacridine on the thermal melting profile of a poly(dA)[poly(dT)]₂triplex were compared 9-Aminoacridine stabilizes the triplex far more effectively than anthryl derivatives. The absorption and CD and LD spectroscopic characteristics of anthryl derivatives are similar to those of 9-aminoacridine when complexed with the triplex; the N atom of acridine, which can act as a hydrogen bond acceptor, plays an important role in triplex stabilization.

http://www.koreascience.or.kr:80/article/JAKO199913464471079.pdf

Stabilization Stabilization of Poly(dA)·[poly(dT)] 2 Triplex by Anthryl and Acridine Derivatives: Effect of Side Chains and Nitrogen Atom on the Polycyclic Aromatic Ring

complex indicates that the extended ligandof the metal complex lie perpendicular to the polynucleotide helix axis. Together with strong hypochromismand red shift in the interligand absorption region, we concluded that the extended BDPPZ or DPPZ ligand in-tercalated between the bases of polynucleotide. The spectral properties of the metal complexes bound to CGC

Binding Mode of (Ruthenium(II)(1,10-Phenanthroline)2L) 2+ to Poly(dG)·poly(dC)·poly(dC) + Triplex DNA

The binding properties of S- and R-ofloxacin to poly[d(A-T) ], poly[d(G-C) ] and poly[d(I-C) ] were studied by circular dichroism 22 2 (CD) and various fluorescence techniques. The spectral properties of R-ofloxacin did not change when it was mixed with poly[d(A-T) ] 2

E binding of S- and R-ofloxacin to various synthetic polynucleotides

Although the transition from B-DNA to the A-form is essential for many biological concerns, the properties of this transition have not been resolved. The B to A equilibrium can be analyzed conveniently because of the significant changes in circular dichroism (CD) and absorption spectrum. CD and linear dichroism (LD) methods were used to examine the binding of water-soluble meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) and its derivatives, Co-TMPyP, with B- and A-calf thymus DNA. B- to A-transitions occurred when the physiological buffer was replaced with a water-ethanol mixture (∼80 v/v %), and the fluorescence emission spectra of TMPyP bound to DNA showed a different pattern under ethanol–water conditions and water alone. The featureless broad emission bands of TMPyP were split into two peaks near at 658 and 715 nm in the presence of DNA under an aqueous solution. In the case of an ethanol–water system, however, the emission bands are split in two peaks near at 648 and 708 nm and 656 and 715 nm...

Comparison of the Binding Geometry of Free-Base and Hexacoordinated Cationic Porphyrins to A- and B-Form DNA

One of the porphyrin moieties of the porphyrin dimer intercalates rapidly between the DNA base-pairs, followed by the slow intercalation of the other. The slow intercalation of the second porphyrin moiety of porphyrin dimer to poly[d(G-C)2] was investigated by normal absorption and circular dichroism spectroscopy. The change in absorbance in the Soret band of porphyrin upon association with poly[d(G-C)2] can be best elucidated by the combination of the two single exponential curves, suggesting the intercalation occurred via two independent first order reactions. Activation energies of these two first order reactions were calculated to be 0.37 kcal/mol and 3.19 kcal/mol, respectively. The intercalation associated with lower activation energy can be assigned to the intercalation of second porphyrin moiety to 5′CG3′ site while that with higher activation energy to 5′GC3′ intercalation site. Increasing the flexibility of poly[d(G-C)2] by adding Na+ ion resulted in an enhancement of the reaction rate for both steps in an exponential manner.

Seog K. Kim

Papers

Stabilization Stabilization of Poly(dA)·[poly(dT)] 2 Triplex by Anthryl and Acridine Derivatives: Effect of Side Chains and Nitrogen Atom on the Polycyclic Aromatic Ring

Binding Mode of (Ruthenium(II)(1,10-Phenanthroline)2L) 2+ to Poly(dG)·poly(dC)·poly(dC) + Triplex DNA

E binding of S- and R-ofloxacin to various synthetic polynucleotides

Comparison of the Binding Geometry of Free-Base and Hexacoordinated Cationic Porphyrins to A- and B-Form DNA

Consecutive intercalation of a cationic porphyrin dimer between the GC base-pairs: a kinetic study