MicroRNA Profiling
MicroRNAs, or miRNAs, are small RNAs that regulate gene expression after transcription has taken place. This occurs in both healthy physiological states and diseased states in many organisms. Due to the range of processes they help regulate, miRNAs can provide valuable biomarkers in disease contexts; this is why profiling miRNA expression is a growing field of study.
microRNA Discovery & Profiling
The Interestin miRNA
miRNAs are short, non-coding RNA fragments that help regulate gene expression. After transcription of the original DNA, the messenger RNA (mRNA) is released and the miRNA can bind to it. This binding promotes degradation of the mRNA or inhibition of translation, thereby hindering protein formation. The miRNAs are few in number compared to mRNA targets, with roughly 1 miRNA for every 30 mRNA. However, these few miRNAs can regulate many mRNAs and therefore have a profound effect on expression.
Given that one miRNA can potentially regulate hundreds of mRNA, it has been inferred that the expression pattern of those hundreds of mRNA can be captured in one single miRNA. This wealth of biological information is appealing to researchers in fields such as developmental biology, tissue differentiation, and disease. miRNA profiling involves the analysis of expression of miRNAs ranging from species level to smaller groups of biological interest.
David Bartel (Whitehead Institute/MIT/HHMI) Part 1: MicroRNAs: Introduction to MicroRNAs
Applications of Profiling
miRNA can regulate which mRNAs get translated into proteins, and therefore can suppress inappropriate gene expression. In developmental biology, the comparison of miRNA profiles can help show which miRNAs are involved in certain developmental processes. Such research has shown that miRNA profiles, or expression patters, can be specific to certain cell types. In addition, miRNAs can halt developmental transitions and help maintain the cell state once it has differentiated. This has led researchers to believe miRNA can have valuable applications in understanding stem cells.
In disease contexts, miRNAs are being explored as sources of biomarkers of certain diseases. miRNA profiles of healthy and diseased tissues are compared to identify the disease biomarkers and potential therapeutic targets. This tactic has been employed in cancer, autoimmune, and neurological diseases.
In particular, abnormal miRNAs are of interest both therapeutically and in studying tumor progression in cancer. The underlying cause for the differences in miRNA expression between healthy and diseased tissues can be due to DNA point mutations, epigenetic variation, or chromosomal changes to miRNA genes. In cancer, miRNA profiling has also been used to clinically diagnose the cancer origin when other identification is difficult. miRNA has even been employed in forensics for similar reasons, as miRNA profiles can help distinguish the types of body fluid found in crime scenes.
Methods of miRNA Profiling
miRNA profiling can be done using a range of methods, such as quantitative reverse transcription polymerase chain reaction (qRT-PCR), miRNA microarray, and RNA sequencing. qRT-PCR involves reverse transcription of the miRNA into cDNA, and subsequent sequencing of the product.
This method is commonly applied to other genetic study and is therefore appealing to laboratories already familiar with the method. To profile miRNA using qRT-PCR, several miRNAs are analyzed in parallel under the same reaction conditions. However, miRNAs can differ drastically in their optimal conditions which means analyzing miRNAs in parallel can be difficult. To overcome this, researchers may use locked nucleic acids (LNAs) in the primers themselves.
Microarrays also analyze several miRNAs in parallel during profiling. In the microarray, miRNAs are fluorescently tagged. DNA-based capture probes are then used to hybridize the tagged miRNA. These probes may or may not contain LNAs, just like in qRT-PCR. The array is then scanned, and the fluorescence is quantified to profile the miRNA. Microarrays allow many more samples to be profiled in tandem, however this method is usually more expensive.
A more novel approach to miRNA profiling is next-generation sequencing. RNA sequencing (RNA-seq) is one such method and is often considered the most powerful new technique for miRNA profiling. Like qRT-PCR, in RNA-seq the sample undergoes reverse transcription to generate a cDNA library. Adaptors are inserted on to the ends of the cDNA strands and the samples are amplified using PCR. The raw sequence data is analyzed using bioinformatics to reveal known miRNAs. Prediction of new miRNAs is also possible.
Further Reading
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