Portelite™ Fluorimetric Quantitation Kits: Companion Reagents Optimized for the CytoCite™ BG100 Spectrofluorometer

Abstract

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Portelite™ Fluorimetric Quantitation Kits, optimized for AAT Bioquest's CytoCite™ BG100 fluorometer, are designed to selectively quantify total nucleic acid, DNA (ssDNA, dsDNA), RNA, endotoxin, and protein, providing more sensitive analysis than comparable UV absorbance-based instrumentation. The aforementioned kits are suitable for a variety of common biological applications, including cloning, sequencing, transfection, qPCR, lipopolysaccharide (LPS) detection, and gel electrophoresis. All assays are performed using the same general protocol, providing a simple mix-and-read assay format. Kits contain concentrated assay reagent, dilution buffer, and pre-diluted standards. The fluorescent probes in each kit have similar spectral properties, being blue light-excitable (480 nm), with emissions between 510 nm and 580 nm. While developed specifically for use with the CytoCite™ BG100 fluorometer, Portelite™ Fluorometric kits may also be used on other commercially available compact fluorometric instruments, such as the Qubit^® fluorometer (Thermo Fisher, Inc.).

Introduction

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Conventional cuvette-based spectrophotometers consume relatively large amounts of precious biological samples. Consequently, compact instruments were devised to address the need to employ biomolecular assays using progressively smaller amounts of sample. For example, the NanoDrop microvolume spectrophotometer (Thermo Scientific) combines fiber optic technology and sample surface tension properties to capture and retain small amounts of material without needing cuvettes or capillaries (Desjardins and Conklin, 2010).

A similar requirement for high-sensitivity fluorescence-based assays, using limited amounts of sample, has led to the development of several compact spectrofluorometers. Unlike the Nanodrop line of compact spectrophotometers which quantify absorbance, measurements performed by the CytoCite™ (AAT Bioquest) and Qubit^® (Thermo Scientific) compact fluorometers rely upon the principle of fluorometry, wherein quantification of as little as 1 mL of DNA, RNA, or protein sample is achieved with high accuracy by employing sensitive fluorescent dyes. Compared with absorbance-based measurements, fluorescence-based measurements exploiting dye-binding are more precise, selective, and sensitive for quantifying different classes of biomolecules (Table 1; Khetan et al., 2019). For instance, optimized intercalating fluorescent dyes have been developed to interact specifically with double-stranded DNA (dsDNA), and thus their quantification is not impacted significantly by contaminating proteins, single-stranded DNA (ssDNA), or RNA molecules.

Absorbance-based methods, as employed by the Nanodrop spectrophotometer, cannot selectively monitor dsDNA, ssDNA, RNA, and nucleotides, as only total sample absorbance is measured. Additionally, with spectrophotometry, nucleic acids are typically measured using their absorbance maxima of 260 nm, while proteins are measured using their absorbance maxima of 280 nm. The ratio of 260 nm-to-280 nm is often employed to monitor sample purity obtained after DNA and RNA extraction (Manchester, 1996). However, inaccurate ratios are often encountered, especially at very low sample concentrations (<10 ng/µL) of nucleic acids, contributing to inaccurate concentration estimates provided by the NanoDrop instrument. This is particularly true, for instance, when monitoring circulating free DNA fragments (cfDNA) released into the blood plasma (Ponti, 2018; Khetan et al., 2019). With fluorometers, such as the Qubit^® and CytoCite™ instruments, deploying fluorescent dyes that are relatively selective for dsDNA, ssDNA, total nucleic acid, RNA, and protein minimizes the impact of any contaminants in the sample that would otherwise produce spurious results during quantification. Successful application of fluorescence-based sample quantification depends upon optimal pairing of a suitable analytical instrument with high-performance reagents.

CytoCite™ BG100 Portable Fluorometer

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The CytoCite™ BG100 instrument is a cloud-integrated, palm-sized fluorometer optimized to detect probes in the ubiquitously employed FITC channel (Ex/Em = 480 nm/520 nm), with an overall limit of detection of as little as 1 nM of fluorescein (Figure 1). For red fluorescence-emitting probes (Ex/Em = 530 nm/620 nm), such as resorufin-based Amplex™ Red and Amplite^® Red peroxidase substrate assays, the CytoCite™ GR100 instrument is recommended. With a small footprint, the CytoCite™ BG100 and CytoCite™ GR 100 portable fluorometers are the most compact fluorometers available on the market, facilitating easy integration into any laboratory, point-of-care use, or field testing environment. As the only commercially available cloud-enabled fluorometer, the CytoCite™ Fluorometer offers several significant advantages relative to other instruments, such as the Qubit^® Fluorometer (Thermo Fisher). Primary differentiating features include:

1. Unlimited storage of sample results
2. Automated daily data backups to prevent unexpected data loss
3. Synchronization of sample results across different analysis platforms, accessible from any authenticated device, such as a smartphone
4. Compatibility with AAT Bioquest's comprehensive Quest Graph™ analytical suite of tools for fast and easy regression modeling, IC50 calculations, and more

The CytoCite™ Fluorometer utilizes sample volumes of as little as 1 uL and is several orders of magnitude more sensitive than absorbance-based instruments, such as the Nanodrop™ (ThermoFisher) for both DNA (absorbance = 260 nm) and protein (absorbance = 280 nm) quantification. It is also compatible with a comprehensive library of bioassays and fluorometric reagents, allowing for rapid, repeatable quantification of small molecules such as calcium, mercury, RNA, DNA, protein, and endotoxin.

Key Applications for Portelite™ Fluorometric Quantitation Kits

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Total Nucleic Acid Detection

The Portelite™ Fluorimetric Total Nucleic Acid Quantitation Kit (17631) measures the total quantity of nucleic acids, including dsDNA, ssDNA, and RNA, in an easy and accurate format. The kit provides all the essential reagents, including Helixyte™ Green All reagent, dilution buffer, and pre-diluted DNA standards. The Helixyte™ Green All reagent (Ex/Em = 509/529 nm) is a sensitive fluorescent nucleic acid probe for measuring the total quantity of nucleic acids in a sample that may contain dsDNA, ssDNA, RNA, and long oligonucleotides. Helixyte™ Green All reagent indiscriminately binds to dsDNA, ssDNA, and RNA. The kit is optimized for measuring the total amounts of nucleic acids using the CytoCite™ or Qubit^® fluorometers.

DNA Detection

In biomedical research laboratories, DNA quantification represents a fundamental procedure required in most molecular biology protocols, including polymerase chain reaction (PCR) amplification and next-generation sequencing (NGS). An assortment of methods is available for DNA quantification, including UV absorbance, agarose gel electrophoresis, and fluorescent DNA-binding dye techniques. The CytoCite™ and Qubit^® instruments are particularly useful for monitoring DNA quality prior to NGS because of their ability to measure intact dsDNA. Importantly, unlike absorbance-based devices, these fluorescence-based systems can readily determine the amount of intact dsDNA, and the quantitative value yielded decreases with the level of fragmentation and denaturation of DNA. Fluorescence spectroscopy is better able than absorbance to evaluate the template activity of DNA for PCR. While qPCR might seem the optimal choice in this context, the procedure is much more time-intensive and expensive than fluorometry.

The Portelite™ High Sensitivity DNA Quantitation Kit (17660, 17661) provides a rapid method to quantify dsDNA using Helixyte™ Green fluorescent dye (Ex/Em = 502/522 nm). The assay is linear over five orders of magnitude of concentration and is highly selective for dsDNA relative to ssDNA and RNA. The kit is designed to be accurate for initial sample concentrations ranging from 1.25 pg/mL to 100 ng/mL. Helixyte™ Green dye exhibits significant fluorescence enhancement upon binding to dsDNA and provides sensitivity that is a few orders of magnitude better than UV absorbance-based assays.

The Portelite™ Fluorimetric DNA Quantitation Kit with Broad Dynamic Range (17665) provides a rapid method to quantify dsDNA with Helixyte™ Green BR dye (Ex/Em = 503/527 nm). This Portelite™ Fluorimetric DNA Quantitation assay is linear over three orders of magnitude. The assay is highly selective for double-stranded DNA (dsDNA) relative to RNA and is optimized for measuring dsDNA concentrations ranging from 10 pg/µL to 10 ng/µL. Helixyte™ Green BR dye exhibits large fluorescence enhancement upon binding to dsDNA and provides sensitivity that is a few orders of magnitude better than UV absorbance-based assays. The abovementioned kits differ with respect to the concentration range of dsDNA assayed, but each is optimized for the CytoCite™ and Qubit^® fluorometers.

Single-stranded DNA plays an essential role in DNA sequencing. In high-throughput sequencing, the sample dsDNA is processed and fragmented. The resulting ssDNA fragments are attached to a glass slide and then base-paired and amplified with a polymerase. The Portelite™ Fluorimetric ssDNA Quantitation Kit (17625) measures single-stranded DNA in an easy and accurate format (Figure 3). This kit includes the Helixyte™ Green ssDNA reagent (Ex/Em = 498/519 nm), dilution buffer, and pre-diluted DNA standards for ~200 tests. Helixyte™ Green ssDNA reagent is a sensitive fluorescent nucleic acid probe for quantifying oligonucleotides and single-stranded DNA (ssDNA) in solution. It can quantify as little as 100 pg/mL oligonucleotide or ssDNA, a sensitivity exceeding absorbance-based methods by more than 10,000-fold. With this kit, as little as 1 ng/mL oligonucleotide or ssDNA can be detected. The Portelite™ ssDNA Quantitation Kit is optimized for quantifying dsDNA using CytoCite™ and Qubit^® fluorometers.

RNA Detection

Detection and quantification of small amounts of RNA are crucial to a wide variety of biomedical applications, including measuring yields of in vitro transcribed RNA and measuring RNA concentrations before performing Northern blot analysis, S1 nuclease assays, RNase protection assays, cDNA library preparation, reverse transcription PCR, and differential display PCR. When analyzing gene expression, the amount of the messenger RNA (mRNA) of a gene reveals the level of gene expression. The most commonly used technique for measuring nucleic acid concentration is the determination of absorbance at 260 nm. The absorbance-based method is limited by the interference caused by proteins, free nucleotides, and other UV-absorbing compounds. The use of sensitive and selective fluorescent nucleic acid stains alleviates this interference problem. Portelite™ Fluorimetric RNA Quantitation Kit (17658, 17659) contains the ultrasensitive StrandBrite™ Green Fluorimetric RNA reagent developed for quantifying RNA in solution. The kit detects as little as 5 ng/mL RNA using a CytoCite™ or QubitÒ fluorometer. The Portelite™ Fluorimetric RNA Quantitation Kit includes our StrandBrite™ Green nucleic acid dye, along with an optimized and robust protocol. It provides a convenient method for quantifying RNA in solution.

Protein Detection

Quantification of protein concentration represents one of the most widely used methods in biomedical research, being particularly applicable to research involving protein extraction, purification, labeling, crystallization, and analysis. Accurate protein quantification is important to a range of other critical assays that require precise results for total protein content in order to generate reliable data. For example, when comparing different protein extracts by SDS-polyacrylamide gel electrophoresis, accurate protein quantitation is necessary to ensure equal amounts of samples are present in each lane. The Portelite™ Fluorimetric Protein Quantitation Kit (11109, 11111) is a rapid and precise fluorescence-based analytical procedure that measures protein concentration in a solution. The assay provides a dynamic range of 12.5 mg/mL to 5 mg/mL when using bovine serum albumin (BSA) as the calibration standard. The kit is more convenient and consumes less precious samples than conventional colorimetric solution assays for protein quantification, such as the bicinchoninic acid (BCA), Bradford (Coomassie Blue), and Lowry assays. The kit is suitable for estimation of protein concentration in measuring protein purity from column fractions after chromatography or samples to be analyzed by electrophoresis, as well as a host of other common research applications. The kit is optimized for performance on the Cytocite™ and Qubit^® fluorometers.

Endotoxin (LPS) Detection

Lipopolysaccharides (LPS), also called endotoxins, are large molecules consisting of a lipid and polysaccharide composed of O-antigen, outer core, and inner core joined by a covalent bond (Dullah and Ongkudon, 2017). Endotoxins are found in the outer membrane of Gram-negative bacteria, and they can bind the CD14/TLR4/MD2 receptor complex in many cell types, particularly in monocytes, dendritic cells, macrophages, and B cells, which subsequently promotes secretion of various pro-inflammatory mediators, leading to septic shock. Detection of contaminating LPS in biopharmaceuticals, particularly protein, peptide, and antibody preparations, is a crucial activity in the context of biomanufacturing and bioprocessing. Portelite™ Rapid Fluorimetric Endotoxin Detection Kit (60008, 60009) uses Endotoxin Green™, a sensitive fluorogenic substrate to detect endotoxin. Endotoxin Green™ is hydrolyzed in the presence of endotoxins and the Limulus Amebocyte Lysate (LAL), an extract of blood cells obtained from horseshoe crabs, to produce a strong green fluorescence signal. The endotoxin activity is proportional to the fluorescence intensity resulting from the hydrolysis of Endotoxin Green™. The kit is optimized for use on Cytocite™ and Qubit^® fluorometers and can detect a broad range of endotoxin (from 1 EU/ml to 0.002 EU/ml) present in biological samples.

Conclusion

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Compact fluorometers, such as AAT BioQuest's CytoCite™ BG100 and Thermo-Fisher's Qubit^® instruments, accurately quantify biological samples, reduce sample consumption requirements and increase assayable dynamic concentration range when compared with conventional cuvette or capillary-based instruments. Compact devices enable the analysis of samples obtained in limited supply, such as material obtained from laser-capture microdissection or needle biopsies. They have also gained popularity when analyzing plentiful sample material due to convenience and minimization of waste disposal requirements. The peak performance of such instruments depends upon the use of optimized fluorescent dyes selective for ssDNA, dsDNA, RNA, total nucleic acid, protein, or endotoxin in order to minimize any impact from sample contaminants on experimental results.

References

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