Absolute Number Of Molecules Research Articles

A novel and robust method for counting components within bio-molecular complexes using fluorescence microscopy and statistical modelling

Cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. Various techniques can provide a qualitative survey of which components are found in a given complex. However, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. Here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. We have devised a system in which batches of a given biomolecule are differentially labelled with spectrally distinct fluorescent dyes (label A or B), and mixed such that B-labelled molecules are vastly outnumbered by those with label A. Complexes, containing this component, are then simply scored as either being positive or negative for label B. The frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. We demonstrate this method using complexes of Adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. Beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology.

TemporalGSSA: A numerically robust R-wrapper to facilitate computation of a metabolite-specific and simulation time-dependent trajectory from stochastic simulation algorithm (SSA)-generated datasets

Whilst data on biochemical networks has increased several-fold, our comprehension of the underlying molecular biology is incomplete and inadequate. Simulation studies permit data collation from disparate time points and the imputed trajectories can provide valuable insights into the molecular biology of complex biochemical systems. Although, stochastic simulations are accurate, each run is an independent event and the data that is generated cannot be directly compared even with identical simulation times. This lack of robustness will preclude a biologically meaningful result for the metabolite(s) of concern and is a significant limitation of this approach. "TemporalGSSA" or temporal Gillespie Stochastic Simulation Algorithm is an R-wrapper which will collate and partition SSA-generated datasets with identical simulation times (trials) into finite sets of linear models (technical replicates). Each such model (time step of a single run, absolute number of molecules for a metabolite) computes several coefficients (slope, intercept, etc.). These coefficients are averaged (mean slope, mean intercept) across all trials of a technical replicate and along with an imputed time step (mean, median, random) is incorporated into a linear regression equation. The solution to this equation is the number of molecules of a metabolite which is used to compute the molar concentration of the metabolite per technical replicate. The summarized (mean, standard deviation) data of this vector of technical replicates is the outcome or numerical estimate of the molar concentration of a metabolite and is dependent on the duration of the simulation. If the SSA-generated dataset comprises runs with differing simulation times, "TemporalGSSA" can compute the time-dependent trajectory of a metabolite provided the trials-per technical replicate constraint is complied with. The algorithms deployed by "TemporalGSSA" are rigorous, have a sound theoretical basis and have contributed meaningfully to our comprehension of the mechanism(s) that drive complex biochemical systems. "TemporalGSSA", is robust, freely accessible and easy to use with several readily testable examples.

Nuclear and Cytoplasmatic Quantification of Unconjugated, Label-Free Locked Nucleic Acid Oligonucleotides.

Methods for the quantification of antisense oligonucleotides (AONs) provide insightful information on biodistribution and intracellular trafficking. However, the established methods have not provided information on the absolute number of molecules in subcellular compartments or about how many AONs are needed for target gene reduction for unconjugated AONs. We have developed a new method for nuclear AON quantification that enables us to determine the absolute number of AONs per nucleus without relying on AON conjugates such as fluorophores that may alter AON distribution. This study describes an alternative and label-free method using subcellular fractionation, nucleus counting, and locked nucleic acid (LNA) sandwich enzyme-linked immunosorbent assay to quantify absolute numbers of oligonucleotides in nuclei. Our findings show compound variability (diversity) by which 247,000–693,000 LNAs/nuclei results in similar target reduction for different compounds. This method can be applied to any antisense drug discovery platform providing information on specific and clinically relevant AONs. Finally, this method can directly compare nuclear entry of AON with target gene knockdown for any compound design and nucleobase sequence, gene target, and phosphorothioate stereochemistry.

Internal rulers to assess fluorescent protein photoactivation efficiency.

Photoactivatable fluorescent proteins (PA-FPs) have been widely used to assess the dynamics of cell biological processes. In addition, PA-FPs enabled single-molecule based super-resolution imaging (photoactivated localization microscopy) and thereby provided unprecedented structural insight. For the lack of tools, however, the fraction of PA-FPs that is, actually being switched on to fluoresce, that is, the photoactivation efficiency, has been difficult to assess. Uncertainty about photoactivation efficiency has hampered an understanding of the absolute amount of PA-FPs, that is, being examined. Here, we present internal rulers to assess photoactivation efficiencies of photoactivatable proteins. These internal rulers comprise a PA-FP that is genetically directly coupled to a spectrally distinct always-on fluorescent protein. Thus, these fluorescent proteins will be expressed in the bacterial and mammalian cell in a one-to-one ratio. With these tools, we describe photoactivation efficiencies of PA-GFP and PA-Cherry in intensity-based ratiometric ensemble studies and on the single-molecule level. In ratiometric ensemble studies, we show that photoactivation efficiency depends on how the PA-FPs are exposed to 405nm light. Using a laser-scanning microscope, hundreds of iterative low-level exposures are up to four times more efficient than a short high-power exposure. Using wide-field illumination, photoactivation was similarly efficient and instantaneous. These findings suggest that the repetitive or stochastic exposure to photons of 405nm light results in more efficient photoactivation than a continuous flow of photons. Because of the differential photoactivation efficiency, it is crucial to assess photoactivation efficiency for any given experimental set-up. The tools we provide can be applied to any genetically encoded photoactivatable protein. Determination of photoactivation efficiency is essential for an understanding of absolute molecule numbers in ensemble studies and, most importantly, quantitative superresolution imaging. © 2017 International Society for Advancement of Cytometry.

The Lifetime NAD(P)H Fingerprint of Salmonella Infection

The Lifetime NAD(P)H Fingerprint of Salmonella Infection

Noise Response of P53 Oscillation against System Size Scaling

p53 is a DNA binding protein regulating the expression of various other proteins under DNA damaging cell stress. Based on different type and extent of DNA damage, p53 up-regulates different sets of proteins and triggers cell cycle arrest, DNA repair or Apoptosis. Given the importance of p53 regarding to its role in cell fate decision, it is only reasonable for such molecules to be under delicate regulation.One interesting observation on p53 dynamics is that when stress signal is activated, rather than staying in an activated higher level, the concentration of p53 pulses with a period of around 5.5 hours, in a rather noisy manner. mdm2, one of p53's main regulators, goes through a similar pulsing process, with a same period as the oscillation of p53 and a stable delay in phase. So far, different theories on both the origin of p53 oscillation and its variation on amplitude are explored. Yet the relation between the size of the system and the noise-driven oscillation, or rather, whether the oscillation is robust against the size variation is hardly mentioned. Given that the absolute number of molecules in a single cell being difficult to assess experimentally, along with the fact that cell size varies for different cell type in a single species, we believe a discussion on how such dynamics behaves under cell size variation being valid for both theoretical and application purposes. In our work, we adopted and modified an existing model. Based on it, we shall discuss its noise driven oscillation behaviour and explore its response against system size variation both numerically and analytically.

Modelling calcium-dependent proteins in the spine - challenges and solutions

Event Abstract Back to Event Modelling calcium-dependent proteins in the spine - challenges and solutions Melanie I. Stefan1*, Shirley Pepke1, Stefan Mihalas1, Thomas Bartol2, Terrence Sejnowki2 and Mary B. Kennedy1 1 California Institute of Technology, Biology Division, United States 2 Salk Institute, United States Synaptic plasticity is mediated by calcium signalling in the postsynaptic spine. One hypothesis suggests that the direction of plasticity is determined by the relative activation of two Ca2+-/calmodulin dependent proteins, CaMKII and calcineurin [Lisman. PNAS, 1989], and the subsequent activation of tightly regulated signalling pathways in the spine. Computational modelling allows us to gain an insight into how proteins within this complex signalling pathway may be regulated and in turn regulate each other. It can also improve understanding of its complex and often non-intuitive behaviour under varying conditions. Three features of this signalling system pose a challenge to computational modellers: First, the absolute number of molecules in a dendritic spine is small, which makes reactions stochastic and affects competition between proteins. Second, the constrained geometry within the dendritic spine affects diffusion and creates distinct, dynamic signalling microdomains. Third, the large number of possible modifications on signalling proteins such as CaMKII increases combinatorial complexity and requires modelling strategies that can work with large numbers of possible states. To manage all three of these challenges, we are using the spatial stochastic simulator MCell [Kerr et al. SIAM J Sci Comput, 2008]. MCell is an agent-based Monte-Carlo simulator that allows stochastic modelling in arbitrarily complex geometries. It is therefore ideally suited to model systems with small molecule numbers and spatial constraints. A realistic reconstruction of a portion of a dendrite has recently been used for modelling calcium transients within CA1 neurons using MCell [Keller et al. submitted]. We are currently combining this technology with an earlier kinetic model of calmodulin activation by calcium [Pepke et al. PLoS Comput Biol, 2010] to explore the relationship between calcium signalling, calmodulin activation and the regulation of calmodulin targets in the spine. As an agent-based simulator, MCell allows for simulation of multistate signalling systems which are so complex as to be intractable using ODE/PDE or the Gillespie algorithm. This allows us to use MCell to construct a detailed model of CaMKII activation that includes all the complexities of calmodulin binding [Stefan et al. PLoS ONE, 2012], phosphorylation [Miller and Kennedy. Cell, 1986], conformational change and intramolecular regulation [Chao et al. Nat Struct Mol Biol, 2010]. Keywords: computational neuroscience, modelling, Calcium signalling, calcium dependent proteins, computational modeling Conference: 5th INCF Congress of Neuroinformatics, Munich, Germany, 10 Sep - 12 Sep, 2012. Presentation Type: Poster Topic: Neuroinformatics Citation: Stefan M, Pepke S, Mihalas S, Bartol T, Sejnowki T and Kennedy M (2014). Modelling calcium-dependent proteins in the spine - challenges and solutions. Front. Neuroinform. Conference Abstract: 5th INCF Congress of Neuroinformatics. doi: 10.3389/conf.fninf.2014.08.00091 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Mar 2013; Published Online: 27 Feb 2014. * Correspondence: Dr. Melanie I. Stefan, California Institute of Technology, Biology Division, Pasadena, United States, mstefan@caltech.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Melanie I. Stefan Shirley Pepke Stefan Mihalas Thomas Bartol Terrence Sejnowki Mary B. Kennedy Google Melanie I. Stefan Shirley Pepke Stefan Mihalas Thomas Bartol Terrence Sejnowki Mary B. Kennedy Google Scholar Melanie I. Stefan Shirley Pepke Stefan Mihalas Thomas Bartol Terrence Sejnowki Mary B. Kennedy PubMed Melanie I. Stefan Shirley Pepke Stefan Mihalas Thomas Bartol Terrence Sejnowki Mary B. Kennedy Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Quantitative Storm using Organic Dyes

Quantitative super-resolution imaging is important for understanding and determining the exact stoichiometry of molecular complexes or oligomerization states of proteins within a cell. This has been done previously using fluorescence proteins, however these proteins require extensive computational correction due to their blinking. Organic dyes are commonly used for super-resolution imaging because of their high photon numbers, unfortunately blinking corrections for these dyes are very difficult. Here, we develop a technique for quantitative super resolution microscopy that allows us to count the absolute number of molecules using organic “caged” dyes, which do not blink. We calibrate our system and find that caged-fluorescein and caged-carborhodamine, when conjugated with SNAP-tag labeling, allow for quantitative counting of targets. We apply this technique to the characterization of the oligomerization states of chemokine receptors in mammalian cells.

Feshbach-optimized photoassociation of ultracold6Li87Rb molecules with short pulses

Two-color photoassociation of ground state $^6$Li$^{87}$Rb molecules via the $\mathrm{B}^1\Pi$ electronic state using short pulses near a magnetic Feshbach resonance is studied theoretically. A near-resonant magnetic field is applied to mix the hyperfine singlet and triplet components of the initial wave function and enhance the photoassociation rate, before the population is transferred to the ground state by a second pulse. We show that an increase of up to three orders of magnitude in the absolute number of molecules produced is attainable for deeply bound vibrational levels. This technique can be generalized to other molecules with accessible magnetic Feshbach resonances.

Modelling Ca2+ -dependent proteins in the spine - challenges and solutions

Background / Purpose: Modelling post-synaptic proteins poses three technical problems: small absolute molecule numbers, large numbers of possible states, and the complex geometry of the spine, which is not a well-mixed compartment. Computational approaches are needed that solve all three of these problems. Main conclusion: Stochastic simulation methods can be used for systems with small molecule numbers, agent-based methods to represent multi-state molecules, and spatial methods to simulate events in complex geometries. We used the agent-based spatial stochastic simulator MCell to model the Ca2+-dependent activation of calmodulin and Ca2+/calmodulin-dependent kinase II (CaMKII) in the spine. Next steps: Next steps will include the extension of our model to include more interaction partners, and to represent some of the regulation events in more detail.

Quantitative Analysis of Fission Yeast Transcriptomes and Proteomes in Proliferating and Quiescent Cells

SummaryData on absolute molecule numbers will empower the modeling, understanding, and comparison of cellular functions and biological systems. We quantified transcriptomes and proteomes in fission yeast during cellular proliferation and quiescence. This rich resource provides the first comprehensive reference for all RNA and most protein concentrations in a eukaryote under two key physiological conditions. The integrated data set supports quantitative biology and affords unique insights into cell regulation. Although mRNAs are typically expressed in a narrow range above 1 copy/cell, most long, noncoding RNAs, except for a distinct subset, are tightly repressed below 1 copy/cell. Cell-cycle-regulated transcription tunes mRNA numbers to phase-specific requirements but can also bring about more switch-like expression. Proteins greatly exceed mRNAs in abundance and dynamic range, and concentrations are regulated to functional demands. Upon transition to quiescence, the proteome changes substantially, but, in stark contrast to mRNAs, proteins do not uniformly decrease but scale with cell volume.

Single-Molecule Counting with Palm

Photoactivated Localization Microscopy (PALM) is a single-molecule based imaging technique. By repeated activation and sampling of sparse subsets, densely expressed fluorescent proteins can be resolved in time. Here, we describe how to exploit this property for single-molecule counting in cells. A strategy is developed for differentially activating and optimized imaging of PA-GFP and PA-Cherry. Coupled PA-FPs are used as calibration probes for standardization of single-molecule counting and determination of photoactivation efficiency. With these tools, we demonstrate how single-molecule counting enables to assess stoichiometries and to relate such counts to absolute molecule numbers in a biological specimen.

Thermal lens detection device

A thermal lens detection device was developed to realize an easy-to-use, portable and sensitive detector for nonfluorescent molecules. Two laser diodes (658 nm for excitation and 785 nm for probe) were made coaxial in an optical unit and were coupled to a single-mode optical fiber. On a microfluidic chip, a small holder for the optical fiber was fixed, and micro-lenses (numerical aperture of 0.2) were also integrated inside the holder. The micro-lenses were designed to realize an adequate chromatic aberration (50 μm), which was essential for sensitive thermal lens detection. Compared with conventional thermal lens detection systems which required very laborious and accurate optical alignment with the microchannel, the new device needed just attachment-detachment of the optical fiber, which was important for practical application. The lower limit of detection was 10 nM for nickel(II) phthalocyaninetetrasulfonic acid tetrasodium salt solutions (model sample), and the absorbance was 9 × 10(-6) AU. The absolute number of molecules detected was less than 200 zmol. The coefficient of variance for 5-time attachment-detachment of the optical probe was as small as 3.6%. The technical development allowed integration of the thermal lens detection devices inside a microsystem (e.g. enzyme-linked immuno-sorbent assay system), and practical microsystems were realized with sensitivities several-orders higher than absorptiometry.

Quantification of DNA and protein adsorption by optical phase shift

A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA. Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to ∼1 ng/mm 2 surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.

Heterodyne single‐beam CARS microscopy

AbstractIn single‐beam coherent anti‐Stokes Raman spectroscopy (CARS), a complete four‐wave‐mixing scheme is accomplished within a single pulse from an ultrashort femtosecond oscillator. Spectral information is achieved by coherent control of the non‐linear signal generation using phase modulation of the excitation with a pulse shaper. This single‐beam approach can be even extended to heterodyne detections schemes wherein a strong local oscillator (LO) field, also generated by pulse shaping from the same excitation laser, interferes with the CARS signal. Here, we explore the potential of this promising technique. A careful optimisation of the experimental scheme is presented together with an analysis of the different relations between LO field and CARS signal intensity, discovering an optimal LO field at a fraction of about 10−4 compared to the overall CARS excitation energy. With the optimised set‐up, an absolute number of molecules as low as 5 × 106 corresponding to concentrations in the attomole regime in the focal volume can be detected. Using such an optimal parameterisation, heterodyne single‐beam CARS based on a broadband laser source is introduced to microscopic applications, combining chemical selectivity and a high spatial resolution of the non‐linear CARS process with the sensitivity of the heterodyne approach. Copyright © 2009 John Wiley & Sons, Ltd.

Photoassociation with chirped laser pulses: calculation of the absolute number of molecules per pulse

The total number of molecules produced in a pulsed photoassociation of ultracold atoms is a crucial link between theory and experiment. A calculation based on first principles can determine the experimental feasibility of a pulsed photoassociation scheme. The calculation method considers an initial thermal ensemble of atoms. This ensemble is first decomposed into a representation of partial spherical waves. The photoassociation dynamics is calculated by solving the multichannel time-dependent Schrödinger equation on a mapped grid. The molecules are primarily assembled in a finite region of internuclear distances, the ‘photoassociation window’. The ensemble average was calculated by adding the contributions from initial scattering states confined to a finite volume. These states are Boltzmann averaged where the partition function is summed numerically. Convergence is obtained for a sufficiently large volume. The results are compared to a thermal averaging procedure based on scaling laws which leads to a single representative initial partial wave which is sufficient to represent the density in the ‘photoassociation window’. For completeness a third high-temperature thermal averaging procedure is described which is based on random phase thermal Gaussian initial states. The absolute number of molecules in the two first calculation methods agree to within experimental error for photoassociation with picosecond pulses for a thermal ensemble of rubidium or caesium atoms in ultracold conditions.

A new concept for ultrasensitive fluorescence measurements of molecules in solution and membrane:: 1. Theory and a first application

Just because there is an average of one molecule in the observation volume of a solution or membrane (single-phase), one cannot say that this is an individual molecule since many different single molecules measured one by one or the same single, individual molecule not leaving the detection volume on time average can cause a single-molecule event. The latter case is of interest and allows the continuous observation of one and the same single molecule without averaging over many ‘different’ single molecules. For the first time a universal theoretical and experimental framework is presented for the continuous observation of the same single, individual molecule without immobilization, hydrodynamic flow, or burst size histograms of fluorescence intensity traces. In this original article, the stochastic approach is derived and its main characteristics are demonstrated with the free fluorophore rhodamine-green in solution for simpler experimental realization. Single (solution)-phase single-molecule fluorescence auto- (or two-color cross-) correlation spectroscopy (SPSM-FCS) is used as a specific application in order to count the absolute number of molecules in the observation volume. The absolute number of molecules, the diffusion coefficient of the single fluorescent molecule, the lower limit of distance, and the molar concentration of the bulk phase (solution) were directly obtained from the measured auto- or (cross)-correlation curves of the SPSM-FCS experiments. For this purpose, the detection volume that was measured was less then 1 fl (10 −15 l). Then, a concentration of the bulk solution was chosen in such a way that the probability of detecting more than one molecule in the detection volume was very small. The Poisson probability was experimentally determined for the absolute number of molecules depending upon a specified bulk concentration. From the diffusion coefficient of the molecule, it was found that the probability of the molecule diffusing out of the probe volume during the measurements was negligibly small.

Transcription profiles of the bacterium Mycoplasma pneumoniae grown at different temperatures.

Applying microarray technology, we have investigated the transcriptome of the small bacterium Mycoplasma pneumoniae grown at three different temperature conditions: 32, 37 and 32 degrees C followed by a heat shock for 15 min at 43 degrees C, before isolating the RNA. From 688 proposed open-reading frames, 676 were investigated and 564 were found to be expressed (P < 0.001; 606 with P < 0.01) and at least 33 (P < 0.001; 77 at P < 0.01) regulated. By quantitative real-time PCR of selected mRNA species, the expression data could be linked to absolute molecule numbers. We found M.pneumoniae to be regulated at the transcriptional level. Forty-seven genes were found to be significantly up-regulated after heat shock (P < 0.01). Among those were the conserved heat shock genes like dnaK, lonA and clpB, but also several genes coding for ribosomal proteins and 10 genes of unassigned functions. In addition, 30 genes were found to be down-regulated under the applied heat shock conditions. Further more, we have compared different methods of cDNA synthesis (random hexamer versus gene-specific primers, different RNA concentrations) and various normalization strategies of the raw microarray data.

Quantitative detection of protein arrays.

We introduce a quantitative method that utilizes scanning electron microscopy for the analysis of protein chips (SEMPC). SEMPC is based upon counting target-coated gold particles interacting specifically with ligands or proteins arrayed on a derivative microscope glass slide by utilizing backscattering electron detection. As model systems, we quantified the interactions of biotin and streptavidin and of an antibody with its cognate hapten. Our method gives quantitative molecule-counting capabilities with an excellent signal-to-noise ratio and demonstrates a broad dynamic range while retaining easy sample preparation and realistic automation capability. Increased sensitivity and dynamic range are achieved in comparison to currently used array detection methods such as fluorescence, with no signal bleaching, affording high reproducibility and compatibility with miniaturization. Thus, our approach facilitates the determination of the absolute number of molecules bound to the chip rather than their relative amounts, as well as the use of smaller samples.

Quantitation of IGF-I, IGF-II, and multiple insulin receptor family member messenger RNAs during embryonic development in rainbow trout.

The IGF system has been shown to be important for normal embryonic growth in mice. Characterization of the IGF system in lower vertebrates is still in progress. To gain a greater understanding of the IGF system during embryonic development in teleosts, a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay was developed and used to quantitate the levels of IGF-I and IGF-II mRNAs from rainbow trout embryos isolated from a staged series. The absolute number of molecules of IGF-I mRNA/microg total RNA was significantly lower than the absolute number of molecules of IGF-II mRNA/microg total RNA both during early and late embryonic development. The recent identification of multiple IGF type I receptor (rtIGFR) and insulin receptor (rtIR) cDNAs in rainbow trout has provided us with a tool to investigate the expression of these mRNAs. A relative quantitative RT-PCR assay was used to determine the steady state levels of two forms of rtIGFR and three forms of rtIR mRNAs in rainbow trout embryos. The relative levels of rtIGFR mRNAs were greater in embryos compared to adult tissues while the relative levels of rtIR mRNAs were generally lower. In a RT-PCR based assay, a differential ability to detect rtIGFR and rtIR mRNAs was shown, suggesting developmental regulation of polyadenylation. Our results suggest that IGF-II mRNA is the predominant IGF expressed in rainbow trout embryos. Our characterization of IGF ligand and receptor mRNA levels in rainbow trout embryos suggests that a functional IGF system exists during embryonic development in teleosts.