Nucleic Acid Detection, Quantification and Imaging

In this issue

Latest News and Events: 69th AACC Annual Scientific Meeting
Buccutite™ Conjugation Kits: Quick and Easy Antibody Labeling
NAADP-AM, A New Player in Calcium Signaling Pathways
Nucleic Acid Detection, Quantification and Imaging
Quantitative Analysis of Thiols and Maleimides

AssayWise Letters 2017, Vol. 6(1)
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Nucleic acid staining is an important tool that gives insight into cellular mechanisms and functionality of macromolecules like DNA and RNA. This is critical as these nucleic acids and their associated organelles are responsible for the development, maintenance and growth of living organisms. The combination of nucleic acid staining with applications such as fluorescence microscopy and flow cytometry also allow for spatiotemporal imaging, providing a powerful means for visualizing stained nucleic components in fixed and live cells.

Image of dead cells stained with Nuclear Blue™ DCS1 (Cat# 17548). Fixed HeLa cells were plated on a 96-well microtiter plate, incubated with 2.5 nM Nuclear Blue™ DCS1 for 230 minutes and imaged with DAPI channel.

Analyzing nucleic acid stains in cells provides an excellent overview of the cellular localization and organization of nucleic macromolecules and organelles with respect to time. Fixed cell stains in tandem with flow cytometry provide a snapshot of the distribution of nucleic macromolecules in a population of cells with respect to a single temporal time point. For example, DNA staining of fixed cells can be used to analyze the relationship between membrane integrity and the degradation of DNA at specific times during cellular events such as apoptosis. Nucleic acid staining in live cells for fluorescence microscopy aids in visualizing and understanding the spatiotemporal dynamics and organization of nucleic acids and organelles in real time. The observation of dynamic changes provides more insight into the operations of nucleic acids as they move within their environment during specific cellular functions, such as the regulation of gene expression. However, differentiating between nucleic macromolecules is considerably challenging and may prove difficult in studies where selective staining of nucleic acids is imperative for spatiotemporal analysis.

Obstacles associated with selectively staining nucleic acids are the structural and chemical similarities shared between DNA and ribonucleic acid (RNA). Both categories of nucleic acids are nearly identical, and therefore many staining techniques are applicable and synonymous for both. This non-specificity for staining nucleic acids can be detrimental to the signal quality of interest because of the impeding background noise from other nucleic molecules. For example, DNA imaging and localization studies may exhibit impeding background interference by RNA because of the non-specific binding of dye-conjugated nucleic acid probes. To address this issue, research has led to the improvement and development of sensitive tools and alternative techniques optimized to specifically target and stain DNA. A simple improvement to DNA staining involves the removal of RNA by pretreating the nucleic acid sample with RNAse, an enzyme that catalyzes the degradation of RNA into smaller components. Some improvements utilize cell-permeable dyes designed to selectively target specific configurations of DNA, such as the minor groove region of double helix DNA. Other improvements combine applications, such as gel electrophoresis and nucleic acid staining, to separate and stain large double-stranded DNA (dsDNA) macromolecules of a biological sample.

The increase in fluorescence intensity of Nuclear Green™ DCS1 (Cat# 17550) with the addition of camptothecin in Jurkat cells. Jurkat cells were treated overnight without (A) or with 20 nM camptothecin (B) in a 37 °C, 5% CO2 incubator, and then dye loaded with Nuclear Green™ DCS1 for 60 minutes. At the end of 15 minutes of Nuclear Green™ DCS1 dye loading, MitoLite™ NIR was added for multicolor analysis. The fluorescence intensity of Nuclear Green™ DCS1 and MitoLite™ NIR was measured with a FACSCalibur (Becton Dickinson) flow cytometer using FL1 channel (Nuclear Green™ DCS1) and FL4 channel (MitoLite™ NIR).

DNA Imaging

Image of a dividing (anaphase stage) human cancer cell taken with an epifluorescence microscope. The cancer cell was fixed and the DNA was stained with DAPI (blue), INCENP stained with GFP (green) and Tubulin stained red.

Deoxyribonucleic acid (DNA) is an organic, molecular complex that carries the genetic instruction for all proteins used for the development, functionality and sustainability of living organisms. DNA is comprised of nucleotide molecules containing one of the four nucleobases — cytosine (C), guanine (G), adenine (A) or thymine (T) - a deoxyribose sugar and a phosphate group. These nucleotides covalently bond with one another to form long nucleic acid sequences that have the capacity to store viable information. In normal conditions, DNA molecules form a double helix consisting of two complimentary DNA strands which run antiparallel alongside each other. To better understand the sensitive mechanisms and functionality of DNA, labeling techniques such as DNA staining have been developed to tag and visualize DNA samples.

DNA imaging of cells can provide insight into the spatiotemporal dynamics of DNA during biological functions. For example, imaging can be used to visualize how the components of DNA move in relationship to one another during cell division. The common classes of commercially available nucleic acids stains are intercalating dyes and minor-groove binders.

Intercalating Dyes and Gel Electrophoresis

DNA molecular weight ladders stained with Cyber Green™ (Cat# 17590) and SYBR^® Green I Nucleic Acid Gel Stain.

Agarose gel electrophoresis is an analytical method that uses an electrical current to separate DNA molecules according to molecular size. Because DNA is colorless, the use of intercalating dyes and loading buffers are combined with electrophoresis for their tagging and fluorescent properties. Intercalating dyes tag DNA for visualization, while the loading buffer increases the density of the DNA sample, impeding DNA solubilization and allowing the sample to be successfully loaded into the wells of the gel.

Ethidium bromide (EtBr) is a commonly used intercalating agent in molecular biology as a fluorescent dye for tagging and visualizing nucleic acids in gel electrophoresis. Its flat structure allows EtBr to intercalate, or insert, between nitrogenous bases in DNA molecules. Upon exposure to UV light, EtBr fluoresces, providing a means to visualize DNA molecules. EtBr's intercalating properties makes it a considerably potent mutagen that can interfere with the functionality of DNA molecules. Because of its high toxicity and mutagenic properties, many labs shy away from using EtBr as a DNA stain. To address toxicity concerns, AAT Bioquest offers a safer set of DNA dyes, Cyber Green™ (Cat# 17590) and Cyber Orange™ (Cat# 17595), that are excellent for nucleic acid gel stains. These dyes are commercially available individually as well as conveniently packaged in AAT Bioquest's Gelite™ Green and Gelite™ Orange Nucleic Acid Gel Staining Kits (Cat# 17589 and Cat# 17594). Gelite™ kits include all the necessary components, proprietary dyes and loading buffers combined with an optimized and robust protocol to effectively visualize nucleic acid gel stains.

Cyber Green™ and Cyber Orange ™ are highly stable and sensitive stains capable of detecting double-stranded DNA (dsDNA) in agarose gels. Their high sensitivity for dsDNA is useful in assays where the presence of RNA or single-stranded DNA (ssDNA) may obscure results. Upon binding to dsDNA, Cyber Green™ emits a fluorescent intensity far greater than EtBr. The quantum yield of the DNA/Cyber Green™ complex is more than 5 times greater than DNA/EtBr complex. The DNA/Cyber Green™ complex has a convenient excitation of 497 nm and emission of 520 nm making it compatible with instruments capable of detecting fluorescein. The DNA/Cyber Orange™ complex has a similar excitation of 495 nm and emission of 540 nm and can also be used in a variety of instrumentations. The major benefit of Cyber Orange™ is its ultrasensitive detectability of dsDNA on gels as little as a picogram. The efficiency and sensitivity of Cyber Green™ and Cyber Orange™ makes them robust alternatives to EtBr for nucleic acid gel staining.

Excitation and Emission of Cyber Green™ Nucleic Acid Gel Stain

Excitation and Emission of Cyber Orange™ Nucleic Acid Gel Stain

The excitation and emission spectra of Cyber Green™ Nucleic Acid Gel Stain (LEFT) and Cyber Orange™ Nucleic Acid Gel Stain (RIGHT) in the presence of calf thymus DNA.

Minor-Groove Binders and Fluorescence Microscopy

The structural significance of double stranded DNA provides another avenue for nucleic acid staining. Double helix DNA molecules have distinct chemical features on their molecular surface that act as recognition sites for small molecules and binding proteins. These sites are characterized by either shallow and wide major grooves, or deep and narrow minor grooves. Transcription factor proteins, essential for the regulation of gene expression, bind to the base pairs of the major grooves in dsDNA. Compounds that bind to the minor grooves in double helix DNA molecules are small fluorescent stains such as DAPI (Cat# 17507) and Hoechst stains (Cat# 17520, Cat# 17530 and Cat# 17537).

Left: Nuclei containing DNA was stained for fluorescence microscopy using Hoechst 33342 (Cat# 17530). Hoechst 33342 can be excited by UV light at 350 nm and will emit cyan fluorescence light with an emission max at 461 nm. Right: DAPI (Cat# 17507), a minor groove binding dye which binds to A-T rich regions of dsDNA, was used to stain and visualize cell nuclei containing DNA.

DAPI (4', 6-diamidino-2phenylindole) and Hoechst stains are cell permeable fluorescent dyes for labelling and visualizing DNA in fluorescence microscopy and flow cytometry. After permeating the cell membrane, these dyes have a strong affinity for A-T rich regions of the minor grooves in DNA. DAPI has an excitation of 358 nm and an emission of 461 nm, and Hoechst stains have a nearly identical excitation of 352 nm and emission of 461 nm. The blue emission of both dyes is convenient for multiplexing assays due to the minimal fluorescent overlap between DAPI or Hoechst stains with traditional green-fluorescent molecules.

Images of HeLa cells stained with Nuclear LCS1 dye series

A composition of fluorescence images of HeLa cells stained with AAT Bioquest's Nuclear LCS1 dye series. HeLa cells were stained with Nuclear Violet™ (17543), Nuclear Green™ (Cat# 17540), Nuclear Red™ (Cat# 17542), Nuclear Yellow (Cat# 17539) and Nuclear Orange™ (Cat# 17541) LCS1 dyes in a Costar black wall/clear bottom 96-well microtiter plate.

AAT Bioquest also offers a comprehensive line of exceptional dyes with various spectral properties that can be combined with other stains for multicolor analysis in fluorescence microscopy. Our Nuclear dye series includes Nuclear Yellow, Nuclear Green™, Nuclear Orange™, Nuclear Red™, and Nuclear Violet™. These dyes are available in cell-permeable forms for staining live cells as well as cell-impermeable forms for staining fixed cells.

Minor groove binders and intercalating dyes, discussed previously, are conventional approaches for imaging and analyzing localization of nucleic acids in cells. Utilization of these dyes in conjunction with imaging and localization studies has confirmed DNA organization and concentration to mitochondrial organelles and to the cell nucleus. For a more comprehensive understanding of the functionality of nucleic macromolecules and organelles, RNA staining is crucial. However, differentiating between RNA and DNA is difficult because of their similar composition. As a result, there has been an increase in interest regarding the research and development of RNA staining techniques. Modifications to dye-conjugated probes and DNA staining techniques can be altered to selectively target and stain cytoplasmic RNA molecules and organelles. Imaging and localization of RNA can provide vital information regarding its functionality in the cellular process of transcription and translation required for protein synthesis.

Additional Information

Table 1. Nucleic Acid Staining Reagents and Assay Kits

Cat No. ▲ ▼	Product Name ▲ ▼	Ex (nm) ▲ ▼	Em (nm) ▲ ▼	Unit Size ▲ ▼
17590	Helixyte™ Green Nucleic Acid Gel Stain 10,000X DMSO Solution	498	522	100 uL
17590	Helixyte™ Gold Nucleic Acid Gel Stain 10,000X DMSO Solution	496	539	1 mL
17507	DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] 10 mM solution in water	358	461	2 mL
17589	Gelite™ Green Nucleic Acid Gel Staining Kit	497	520	1 Kit
17594	Gelite™ Orange Nucleic Acid Gel Staining Kit	497	520	1 Kit
17520	Hoechst 33258 CAS 23491-45-4	352	461	100 mg
17530	Hoechst 33342 Ultrapure Grade	350	461	100 mg
17537	Hoechst 34580 CAS 911004-45-0	368	437	5 mg
17548	Nuclear Blue™ DCS1 5 mM DMSO Solution	350	461	0.5 mL
17550	Nuclear Green™ DCS1 5 mM DMSO Solution	503	526	0.5 mL
17540	Nuclear Green™ LCS1 5 mM DMSO Solution	503	526	0.5 mL
17551	Nuclear Orange™ DCS1 5 mM DMSO Solution	528	576	0.5 mL
17541	Nuclear Orange™ LCS1 5 mM DMSO Solution	514	555	0.5 mL
17552	Nuclear Red™ DCS1 5 mM DMSO Solution	642	660	0.5 mL
17542	Nuclear Red™ LCS1 5 mM DMSO Solution	622	645	0.5 mL
17543	Nuclear Violet™ LCS1 5 mM DMSO Solution	401	459	0.5 mL
17539	Nuclear Yellow [Hoechst S769121] CAS 74681-68-8	355	495	25 mg

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