Accurate RNA quantification is one of the most important steps in molecular biology as it influences the success of downstream applications. Optical spectroscopy is used in most laboratories to quantify and assess RNA concentration and purity quickly and non-destructively. Most molecular biologists will know the expected ratio values to look for that indicate sample purity, though what those numbers actually mean is often overlooked.

This article takes a look at the science behind optical spectroscopy to give researchers a better understanding of their data outputs, as well as other applications of UV-Vis spectroscopy within RNA sequencing workflows.

What is Optical Spectroscopy?

Optical spectroscopy is an analytical technique that uses light to assess samples and materials. It uses light in the UVA, visible, and infrared regions of the electromagnetic spectrum, encompassing wavelengths from 190 to 1000 nm.

To perform optical spectroscopy, a light source is shone at a sample and the way that light interacts with this sample is measured by a spectrometer. The optical spectrometer outputs the measured light intensity as a function of wavelength, and this can be used to gain important information about the sample including its structure.

A typical spectroscopy setup is therefore quite simple and consists of a spectrometer, a sample (which is held within a cuvette holder or a transmission holder) and a light source. The light source that is used may be monochromatic or a broadband source depending on what type of measurement you wish to take. Generally, a monochromatic source is used to excite materials whereas a broadband source is used for absorption, transmission, and reflection measurements.

Jablonski Diagrams Explained

Jablonski diagrams are a written representation of the possibilities that could happen when photons of light interact with a particular molecule. These diagrams are essentially energy diagrams, in which the vertical axis represents the amount of energy and the horizontal axis demonstrates multiplicity. As a result, Jablonski diagrams are commonly used within the field of optical spectroscopy to illustrate electronic and vibrational states, as well as the transitions between them.

Within the diagrams, straight arrows are used to show radiative transitions such as absorption and fluorescence. Wavy arrows, on the other hand, represent non-radiative transitions such as internal conversion and intersystem crossings.

Types of Optical Spectroscopy

Transmittance

Transmittance spectroscopy allows you to quantify how opaque a sample is to a specified wavelength of ligh.

The experiment is performed by shining light from a broadband light onto a sample within a cuvette. The light that is able to pass through is then detected by an optical spectrometer. Transmittance spectroscopy is often used to monitor cell and bacteria growth within industry. The less light that is able to pass through, the higher the cell or bacteria count.

Absorbance

Absorbance is one of most commonly used optical spectroscopy techniques in RNA sequencing workflows. It measures the attenuation of light as it passes through a sample and provides information that can be used to determine the structure of the sample along with its volume concentration, film thickness, and molar concentration.

It works on the principle of excitation. When light hits a material, the energy of the photons within it can be transferred to the electrons within the material, causing them to be excited into higher energy states. In other words, the electrons within the material absorb the incident light. In optically active materials, UV and visible photons will promote electrons to higher energy electronic orbitals.

Absorbance is the form of optical spectroscopy that is used within RNA sequencing workflows to quantify and assess the purity of extracted RNA without the need for additional reagents. Additionally, absorbance spectroscopy has broad applications in biological analytical science, and one of its most common applications is within pharmaceutical analysis. Individual pharmaceutical compounds can be identified by resolving overlapping absorbance peaks on a UV-Vis spectra.

Photoluminescence

As the name suggests, photoluminescence (PL) spectroscopy measures the photoluminescence of a sample. This is when a sample absorbs photons from the light that is shone onto it, then emits this energy in the form of another, lower-energy photon. PL spectroscopy therefore requires photon absorption.

Fluorescence is a form of photoluminescence that is used in techniques for studying biological samples. It involves the relaxation of an electron through a singlet-singlet transition which means that once an electron absorbs a photon, it almost immediately relaxes and emits another, lower energy photon. This happens within nanoseconds, meaning that samples are only fluorescent whilst light is actively being shone onto them.

PL spectroscopy is commonly used for studying biological samples. For example PL-based fluorescent probes can be used to detect specific RNA sequences before the sequencing workflow begins. PL spectroscopy can also be used to monitor interactions between RNA and DNA or proteins.

What do the 260/280 and 260/230 absorbance ratios tell us in RNA analysis?

Absorbance optical spectroscopy is commonly used to assess the purity of RNA and DNA after extraction. Sample purity is important for obtaining accurate results during analysis, hence optical spectroscopy can be used to highlight whether further purifying steps need to be taken to prevent valuable samples from being wasted.

When used in an optical spectrometer, RNA, dsDNA, ssDNA, and nucleotides will show absorbance peaks at 260nm. Common contaminants, such as proteins and phenol, have an absorbance peak at 280nm. The amount of light that is absorbed at these wavelengths is directly proportional to the concentration of the molecule that is being detected. Optical spectrometers are used to automatically determine the concentration of nucleotides or contaminants present using Beer’s Law, and they are capable of doing so with low sample volumes and without the need for additional reagents.

Based on this principle, the ratio of the absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA.

• A 260:280 ratio of ~1.8 is generally accepted as ‘pure’ for DNA.
• A 260:280 ratio of ~2.0 is generally accepted as ‘pure’ for RNA.

If the measured absorbance ratios vary from this, it could indicate the presence of contaminants.

Interestingly, the 260/280 ratios for the 5 nucleotide bases that comprise DNA and RNA vary widely. When measured independently, the 260/280 ratio for each nucleotide is as follows:

• Adenine (A): 4.50
• Cytosine (C): 1.51
• Guanine (G): 1.15
• Thymine (T): 1.47 (DNA only)
• Uracil (U): 4.00  (RNA only)

This means that the resulting 260:280 ratio for the nucleic acid that is being studied is actually an approximation of the weighted average of the 260/280 ratios for all four of the nucleotides that are present. Therefore, the accepted purity ratios of 1.8 and 2.0 will vary depending on the composition of the nucleic acid that is being analysed. This is also the reason why RNA has a higher accepted purity ratio than DNA, due to the higher 260:280 absorbance ratio of Uracil compared to Thymine.

The 260:230 absorbance ratio is used as a secondary measure of nucleic acid purity, but the principle works the same. In this case, contaminants that have an absorbance peak at 230nm are being assessed for, which includes phenol (particularly TRIzol), EDTA, guanidine HCL, and carbohydrates. As many of these reagents are used in the extraction process it can be particularly useful to check for them in the final nucleic acid solution.

The principle here is the same, but the accepted purity ratios are slightly shifted. For both DNA and RNA, the expected 230:260 ratios typically lie in the 2.0-2.2 range. If the ratio is lower than this, it is usually an indication of the presence of contaminants.

Other uses of Optical Spectroscopy in RNA sequencing workflows

Targeted RNA delivery

Optical spectroscopy can be used within the field of targeted RNA delivery to track RNA localization, monitor enzymatic modifications, and study RNA interactions in biological systems. For example, gold nanoparticle conjugates can be used for nucleic acid delivery. These particles have an absorbance region of 520-550 nm, meaning that a UV-Vis spectrometer can be used to quantify nanoparticle conjugation efficiency and detect them at their intended target area.

Protein contamination detection

Protein contamination within nucleic acid solutions can also be detected using total protein colorimetric assays such as Bradford or BCA assays. These assays rely on chemistry that results in a color change if proteins are present in a solution. This typically involves either the detection of reduced copper from protein-copper chelation or protein-dye binding resulting in a colour change that can be quantified. Absorbance optical spectroscopy is used to detect this colour change, with absorbance signals peaking at 562 nm and 595 nm for the BCA and Bradford assays respectively.

Checking for buffer purity

Optical spectroscopy can be used to troubleshoot unexpected results in the RNA extraction process by assessing the purity of any solutions and reagents that are used. For example, it’s possible to check for contaminants within Tris buffers, wash solutions, and phenol to determine whether they are the cause of any issues, and provide confidence about the reliability of the extraction process.