Mikael Kubista

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

BACKGROUND Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a readers ability to evaluate critically the quality of the results presented or to repeat the experiments. CONTENT The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. SUMMARY Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

Absorption and fluorescence properties of fluorescein

We have characterized the protolytic equilibria of fluorescein and determined the spectroscopic properties of its protolytic forms. The protolytic constants relating the chemical activities (which at low ionic strength equal concentrations) of the cation, neutral form, anion and dianion are pK1 = 2.08, pK2 = 4.31, and pK3 = 6.43. All forms have rather high molar absorptivities being e437FH31 = 53 000, e434FH2 = 11 000, e453FH− = 29 000 (e472FH = 29 000) and e490F2 = 76 900 M−1 cm−1 for the cation, neutral form, anion and dianion, respectively. The dianion has the most intense fluorescence with a quantum yield of 0.93 but also the anion shows considerable fluorescence with a quantum yield of 0.37. The neutral and cationic species are upon excitation converted into the anion and fluoresce with quantum yields of about 0.30 and 0.18, respectively.

Protective role of reactive astrocytes in brain ischemia

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP−/−Vim−/− mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP−/−Vim−/− than in wild-type (WT) mice; GFAP−/−, Vim−/− and WT mice had the same infarct volume. Endothelin B receptor (ETBR) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP−/−Vim−/− astrocytes. In WT astrocytes, ETBR colocalized extensively with bundles of IFs. GFAP−/−Vim−/− astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP−/−Vim−/− than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ETBR-mediated control of gap junctions, and PAI-1 expression.

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.

The interactions between the fluorescent dye thiazole orange and DNA

The interaction of the fluorescent dye thiazole orange (TO) with nucleic acids is characterized. It is found that TO binds with highest affinity to double-stranded (ds) DNA [log (K) approximately 5.5 at 100 mM salt], about 5-10 times weaker to single-stranded polypurines, and further 10-1000 times weaker to single-stranded polypyrimidines. TO binds as a monomer to dsDNAs and poly(dA), both as a monomer and as a dimer to poly(dG) and mainly as a dimer to poly(dC) and poly(dT). The fluorescence quantum yield of TO free in solution is about 2 x 10(-4), and it increases to about 0.1 when bound to dsDNA or to poly(dA), and to about 0.4 when bound to poly(dG). Estimated quantum yields of TO bound to poly(dC) and poly(dT) are about 0.06 and 0.01, respectively. The quantum yield of bound TO depends on temperature and decreases about threefold between 5 and 50 degrees C.

Linear dichroism spectroscopy of nucleic acids.

This review will consider solution studies of structure and interactions of DNA and DNA complexes using linear dichroism spectroscopy, with emphasis on the technique of orientation by flow. The theoretical and experimental background to be given may serve, in addition, as a general introduction into the state of the art of linear dichroism spectroscopy, particularly as it is applied to biophysical problems.

Experimental correction for the inner-filter effect in fluorescence spectra

Recorded fluorescence intensity is in general not proportional to sample concentration owing to absorption of the incident and emitted light passing through the sample to and from the point inside the cell where the emission is detected. This well known inner-filter effect depends on sample absorption and on instrument geometry, and is usually significant even in samples with rather low absorption (the error is about 8% at an absorbance of 0.06 in a 1 cm square cell). In this work we show that a particular experimental set-up can be calibrated for the inner-filter effect from the absorption and fluorescence excitation spectra of a suitable standard. The calibration takes only a few minutes and provides correction with sufficient accuracy for most practical situations.

UV-Vis spectroscopic and chemometric study on the aggregation of ionic dyes in water

The monomer-dimer equilibrium in several ionic dyes (Methylene Blue, Acridine Orange, Nile Blue A, Neutral Red, Rhodamine 6G and Safranine O) has been investigated by means of UV-Vis spectroscopy. The data have been processed by a recently developed method for quantitative analysis of undefined mixtures, based on simultaneous resolution of the overlapping bands in the whole set of absorption spectra. In the cases of Acridine Orange a second chemometric approach has been used as a reference. It is based on a decomposition of the recorded spectra into a product of target and projection matrices using non iterative partial least squares (NIPALS). The matrices are then rotated to give the correct concentrations, spectral profiles of the components and the equilibrium constant. The dimeric constants determined by the two methods were in excellent agreement, evidencing the accuracy of the analysis. From the calculated dimeric constant and monomer and dimer spectra, the structures of the dimeric forms of the studied dyes are estimated.

The need for transparency and good practices in the qPCR literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Quantitative Real-Time PCR Method for Detection of B-Lymphocyte Monoclonality by Comparison of κ and λ Immunoglobulin Light Chain Expression

Background: An abnormal IgL:IgL ratio has long been used as a clinical criterion for non-Hodgkin B-cell lymphomas. As a first step toward a quantitative realtime PCR-based multimarker diagnostic analysis of lymphomas, we have developed a method for determination of IgL:IgL ratio in clinical samples. Methods: Light-up probe-based real-time PCR was used to quantify IgL and IgL cDNA from 32 clinical samples. The samples were also investigated by routine immunohistochemical analysis and flow cytometry. Results: Of 32 suspected non-Hodgkin lymphoma samples analyzed, 28 were correctly assigned from real-time PCR measurements assuming invariant PCR efficiencies in the biological samples. Four samples were false negatives. One was a T-cell lymphoma, one was a diffuse large B-cell lymphoma, and one was reanalyzed and found lymphoma-positive by in situ calibration, which takes into account sample-specific PCR inhibition. Twelve of the samples were fine-needle aspirates, and these were all correctly assigned. Conclusions: This work is a first step toward analyzing clinical samples by quantitative light-up probe-based real-time PCR. Quantitative real-time PCR appears suitable for high-throughput testing of cancers by measuring expression of tumor markers in fine-needle aspirates. The rapid expansion in knowledge of the human genome and the development of techniques for analysis of nucleic acids have opened new possibilities for diagnostics. In our first attempt to use quantitative real-time PCR (Q-PCR) 5 for detection of malignant tumors, we have focused on the analysis of non-Hodgkin lymphomas (NHLs).