Gene expression is the conversion of heritable, genetic information into RNA or protein. Differences and changes in gene expression are important measures for understanding biological systems, including normal development and disease progression.
Gene expression is the process of using information encoded in genes to synthesize a functional gene product. Genes are transcribed into RNA, which is either translated into protein or not. Untranslated RNAs serve as functional RNA end products, such as non-coding RNAs (ncRNAs), long, non-coding RNAs (lncRNAs), cellular RNA catalysts such as rRNAs or tRNAs, or small non-coding RNAs that control gene expression, such as microRNAs or short-interfering RNAs (siRNAs).
Whether an RNA is translated into protein or used directly in the cell, RNA transcript levels are generally equated as a measure of gene expression, and therefore, gene expression analysis typically quantifies either messenger RNA (mRNA) levels or non-translated, functional RNA levels as a first step towards protein expression.
Linking the expression of specific genes to a biological process or phenotype helps scientists understand gene function, biological pathways, and the genes that regulate development, cell behavior, cell signaling, and disease. The specific gene expression patterns or “gene signatures” associated with a biological state can serve as biomarkers for that condition. When researching disease causes, gene expression can be used in identification and assessment of its severity. Similarly, determining changes in gene activity resulting from specific environmental and physical changes can provide insights. Such evaluation helps medical professionals understand the impact of medications and facilitates research towards developing more effective drugs. Likewise, gene expression analysis can be applied to other fields other than medical research. Gene expression can identify environmental conditions under which specific crops are induced to thrive, become susceptible to disease or pests, or environmental conditions that stress crop growth and development.
Measuring gene expression has traditionally involved isolating an intact RNA fraction from samples, immobilizing it, and detecting and quantifying the RNA transcripts of interest. This is usually done using a transcript-specific, labeled probe. Gene expression techniques that use this approach have been limited by their ability to study only a few transcripts per experiment. These techniques include: northern blotting, dot blotting, ribonuclease protection assays (RPAs), serial analysis of gene expression (SAGE), and differential or subtractive hybridization.
Current approaches provide greater detection efficiency than immobilized RNA techniques, and they are adaptable for increased target and sample numbers. They usually involve adding multiple probes to an RNA fraction or directly to a cell lysate. These techniques include quantitative PCR (qPCR), digital PCR (dPCR), next generation sequencing (NGS), microarrays and panels, and in situ hybridization, including fluorescent in situ hybridization (FISH).
One of the most widely used methods for analyzing gene expression is qPCR, also called real-time PCR. qPCR is performed following cDNA synthesis. qPCR uses a primer pair like a typical PCR reaction with a fluorescent reporter oligonucleotide (probe) or intercalating dye, to measure amplification of the target sequence during PCR temperature cycling.
IDT offers a selection of qPCR reagents to assess gene expression. Order ready-to-use PrimeTime™ qPCR Assays or design a custom assay. Both probe-based and intercalating-dye based assay options are available.
Familiarize yourself with critical parameters of qPCR assay design, specificity analysis, data interpretation, and troubleshooting. This complete 62-page guide covers every aspect of probe-based qPCR assays.
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