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Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR
Published on: January 22, 2014
Cerebrospinal fluid MicroRNA profiling using quantitative real time PCR
Marco Pacifici1, Serena Delbue, Ferdous Kadri
1Medical School and Stanley S. Scott Cancer Center, LSU Health Sciences Center.
Abstract:
MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis. There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software. Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.
Insights
This study presents a sensitive quantitative real-time PCR method for profiling microRNAs (miRNAs) in cerebrospinal fluid. This technique enables the detection of miRNA expression profiles for potential biomarker discovery in neurological diseases.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) are key regulators of gene expression, influencing translational repression of messenger RNAs (mRNAs).
- Altered miRNA expression is implicated in complex diseases, and circulating miRNAs in body fluids show stability and potential as biomarkers.
- Cerebrospinal fluid (CSF) miRNA profiling offers a promising avenue for identifying disease-specific signatures.
Purpose of the Study:
- To describe a sensitive quantitative real-time PCR (qPCR) method for profiling microRNAs (miRNAs) in cerebrospinal fluid (CSF).
- To establish a reliable protocol for miRNA detection in various biological samples, including CSF, for biomarker analysis.
Main Methods:
- Utilized Exiqon microRNA ready-to-use PCR human panels (I and II V2.R) for detecting 742 unique human miRNAs.
- Performed quantitative real-time PCR (qPCR) assays in triplicate runs.
- Processed and analyzed data using GenEx Professional 5 software.
Main Results:
- Successfully established and validated a sensitive qPCR protocol for miRNA profiling.
- Demonstrated the protocol's efficacy across diverse sample types: cell lines, primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.
- The method allows for the detection of a broad range of human miRNAs.
Conclusions:
- Quantitative real-time PCR provides a sensitive and effective method for profiling microRNAs in cerebrospinal fluid.
- This validated protocol supports the identification of miRNA expression profiles in CSF, crucial for biomarker discovery in neurodegenerative disorders and other conditions.
- The protocol's versatility extends to various sample types, enhancing its utility in biomedical research.
