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Quantitation of nucleic acids is an essential step in sample preparation that ensures the best results from downstream experiments. A prevalent misunderstanding is that all quantitation methods are equivalent in terms of accuracy. However, depending on the approach employed, concentration estimates might vary.
With the increasing range and capabilities of sophisticated assays currently available to scientists, genomics research is becoming constrained less by molecular biology techniques and more by the questions that they are addressing. Continued advancements in qPCR, microarrays and sequencing have aided progress in applications such as genotyping, copy number variation (CNV) analysis, pathogen identification, gene expression analysis, biomarker development, and cytogenetic and molecular oncology profiling.
Various components might hamper the performance and repeatability of these downstream tests in the sample preparation processes. Variation may be introduced in various ways, from sample gathering and storage through nucleic acid extraction and quality control. This blog focuses on a vital but frequently overlooked part of quality control:
Even commonplace technology, such as real-time PCR, might generate inconsistent results, making it difficult to draw inferences from a given experiment. In numerous aspects, quantifying nucleic acid samples is critical to the effectiveness of these assays:
Ensure linear amplification
A delicate balance of nucleic acid template, primers, probes, and other master mix components, as well as cycling settings, is required for real-time PCR reactions. Concatamers, primer dimers, and other non-specific artefacts are minimised with accurate nucleic acid quantification, ensuring linear amplification of target amplicons.Consideration of amplification inhibitors
The efficiency of nucleic acid purification extraction and sample purity is affected by several factors, including sample type and extraction technique. Co-purified pollutants are ubiquitous in many circumstances and can function as amplification inhibitors. It's critical to quantify your sample because samples with the highest concentration of template also contain the highest amount of inhibitors, which might result in a delayed Cq. Samples with lower template concentrations, on the other hand, have lower inhibitor levels, therefore the Cq is slightly delayed.Normalise samples for gene expression applications
Your gene of interest must be standardised to one or more reference genes when comparing gene expression. When you quantify nucleic acid, you can be sure that you're running the same amount of reference and sample simultaneously.Ensure sufficient RNA input
Variation in the amount of RNA utilised in the RT stage might cause difficulties with reproducibility in molecular approaches that require cDNA. Low mRNA input can also cause variability in next-generation sequencing owing to poor amplification. This ineffective amplification of low-to-moderately expressed transcripts may obscure modest biological differences. It's critical to quantify RNA before amplification to ensure that enough RNA is utilised in the test.Methods for Quantitating Nucleic Acid
The concentration of nucleic acids in a sample can be estimated using a variety of scientific procedures. However, a widespread misunderstanding is that all of these procedures are equally accurate or even measure the same thing: the amount of DNA or RNA in the sample. Every approach, in reality, measures something different.Spectrophotometry
The Beer-Lambert Law is a relationship between light absorption and the qualities of the substance through which it travels. The transmission of light through a substance has a logarithmic relationship with the product of the substance's absorption coefficient and the route length, according to this law. The heterocyclic rings of nucleotides (adenine, guanine, cytosine, and thymine/uracil) cause nucleic acid molecules to absorb UV light maximum at 260nm (max = 260nm) in DNA and RNA.Fluorescence
Fluorometry, often known as spectrofluorometry (or just fluorescence), is a sort of electromagnetic spectroscopy that examines the light emitted by fluorogenic molecules known as fluorophores. When compared to other molecules, fluorophores respond differently to light. When an electron of a fluorophore absorbs a photon of excitation light, the electron's energy level increases to an excited state. Some of the energy is wasted during the brief excitation phase, and the remainder is released as a photon to relax the electron back to its ground state. The emitted fluorescence can be separated from the excitation light with a fluorometer because the emitted photon generally contains less energy and hence has a longer wavelength than the excitation photon.Real-Time PCR (qPCR)
Real-time PCR (also known as qPCR) is a technology that uses thermal cycling to melt DNA and enzymatically replicate selected amplicons by DNA polymerase. A total of 2n PCR products are generated after n cycles of heat cycling. After each round of PCR amplification employing fluorescence detection devices, the amount of DNA product is measured. Detection equipment uses fluorescent reporters to assess the buildup of DNA product after each round of PCR amplification. Dyes like SYBR® green or probes like TaqMan® can be used as reporters. Following an initial, according to this law, the reverse transcription (RT) step to convert RNA to cDNA, the same procedure may be used to quantify RNA (Blatter, 2018).You can get a DNA extraction kit and RNA extraction kit at MBP Inc. Order now!
Bibliography Blatter, A. (2018). Choosing the Right Method for Nucleic Acid Quantitation. Promega Global. https://worldwide.promega.com/resources/pubhub/choosing-the-right-method-for-nucleic-acid-quantitation/#abstract-88000b3d-fb67-4354-abb1-381c79553eed
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