Robert Sjöback

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.

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.

Characterization of fluorescein-oligonucleotide conjugates and measurement of local electrostatic potential.

The properties of fluorescein are substantially altered upon conjugation to nucleic acids, affecting not only the molar absorptivities and fluorescence quantum yields but also the protolytic equilibrium constant and fluorescence lifetimes. Around neutral pH, the fluorescein moiety is present as both mono- and dianion, and the pKa relating them is increased from 6.43 for free fluorescein to about 6.90 for fluorescein attached to both single- and double-stranded oligonucleotides of at least 12 bases/base pairs. This difference reflects the local electrostatic potential around the nucleic acid, which is calculated to -28 mV. The molar absorptivities and spectral responses of the conjugated fluorescein protolytic species are also determined, from which the concentrations of fluorescein-oligonucleotide conjugates can be calculated by assuming: epsilon 494 = 62000/[1 + 10-(pH-6.90)] + 12000/[1 + 10(pH-6.90)] (M-1 cm-1). The fluorescence quantum yield of the conjugates depends, in a complex way, on temperature, environment and oligonucleotide length, sequence and conformation, and must be determined for each experimental situation.

DNA Tetraplex Formation Studied with Fluorescence Resonance Energy Transfer

It is emerging that DNA tetraplexes are pivotal for many major cellular processes, and techniques that assess their structure and nature to the point are under development. Here we show how the structural conversion of largely unstructured single-stranded DNA molecules into compact intrastrand DNA tetraplexes can be monitored by fluorescence resonance energy transfer. We recently reported that intrastrand tetraplex formation takes place in a nuclease hypersensitive element upstream of the human c-myc proto-oncogene. Despite the highly repetitive guanine-rich sequence of the hypersensitive element, fluorescence resonance energy transfer measurements indicate that only one well defined tetraplex structure forms therein. The proposed structure, which is specifically stabilized by potassium ions in vitro, has a core of three stacked guanine tetrads that is capped by two intrastrand A-T base pairs.

Quantitative spectral analysis of multicomponent equilibria

Abstract A generalization is presented of a method for the determination of equilibrium constants for binary mixtures. It is shown that titrations involving several components can be quantitatively characterized in terms of component concentrations and spectral responses, even when none of the component spectra is known and the components cannot be obtained in pure form. The method is efficient even if the component spectra overlap extensively. It is applicable to all spectroscopic techniques that provide a linear response, opening up new possibilities to analyze complex chemical systems. The approach is illustrated by determining the absorption spectra and protolytic constants for the fluorescein protolytic equilibria.

MAKING REFERENCE SAMPLES REDUNDANT

Chemometric methods to analyze spectroscopic data without using reference spectra are discussed. The data are first decomposed into principal components, and the number of contributing species is determined by statistical tests. The principal components are then rotated to produce spectroscopic responses and concentration profiles of the chemical species present. Samples that vary in a physical property like pH, total concentration, temperature, or ionic strength, are analyzed by regular 1-dimensional spectroscopy assuming that the components are in chemical equilibrium. Samples containing noninteracting compounds are analyzed by multidimensional spectroscopy, and the principal components are calculated by Procrustes rotation. Several applications of the two approaches on absorption and fluorescence data are presented.

Direct Cell Lysis for Single-Cell Gene Expression Profiling

The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA) to be the best lysis agent, resulting in efficient cell lysis, high RNA stability, and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single-cells as well as samples composed of small numbers of cells.

Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR)

Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.

Gene Expression Signatures in Circulating Tumor Cells Correlate with Response to Therapy in Metastatic Breast Cancer

BACKGROUND Circulating tumor cells (CTCs) are thought to be an ideal surrogate marker to monitor disease progression in metastatic breast cancer (MBC). We investigated the prediction of treatment response in CTCs of MBC patients on the basis of the expression of 46 genes. METHODS From 45 MBC patients and 20 healthy donors (HD), 2 × 5 mL of blood was collected at the time of disease progression (TP0) and at 2 consecutive clinical staging time points (TP1 and TP2) to proceed with the AdnaTest EMT-2/StemCellSelectTM (QIAGEN). Patients were grouped into (a) responder (R) and non-responder (NR) at TP1 and (b) overall responder (OR) and overall non-responder (ONR) at TP2. A 46-gene PCR assay was used for preamplification and high-throughput gene expression profiling. Data were analyzed by use of GenEx (MultiD) and SAS. RESULTS The CTC positivity was defined by the four-gene signature (EPCAM, KRT19, MUC1, ERBB2 positivity). Fourteen genes were identified as significantly differentially expressed between CTC+ and CTC- patients (KRT19, FLT1, EGFR, EPCAM, GZMM, PGR, CD24, KIT, PLAU, ALDH1A1, CTSD, MKI67, TWIST1, and ERBB2). KRT19 was highly expressed in CTC+ patients and ADAM17 in the NR at TP1. A significant differential expression of 4 genes (KRT19, EPCAM, CDH1, and SCGB2A2) was observed between OR and ONR when stratifying the samples into CTC+ or CTC-. CONCLUSIONS ADAM17 could be a key marker in distinguishing R from NR, and KRT19 was powerful in identifying CTCs.