Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Summary

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

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.

Introduction

MicroRNAs belong to the family of small (21-23 nt in length) noncoding RNAs that regulate gene expression post-transcriptionally. microRNAs can be secreted in the extracellular space where they appear to be relatively stable. While determining changes in miRNA expression can be an important step toward identification of biomarkers, performing miRNA profiles and handling a large amount of data may be intimidating.

Here, we describe a protocol to determine changes in miRNA expression in the cerebrospinal fluid (applicable to other body fluids) by a sensitive real time PCR. We also describe the use of software for data analysis, including stat....

Protocol

The following protocol describes the standard procedure to isolate RNA from CSF and profile microRNAs using ready PCR plates. Note that the organic phase extraction is optional, and that a carrier RNA is added to the sample during extraction ensuring maximal recovery of RNA. Consequently, there is no need to quantify the RNA.

Overall, an average of 6-8 plates can be run in one day if the cDNA is synthesized the day before (about 2 hr). Analysis of data is performed when all samples have been profiled and loaded into the software. Depending on the number of samples/groups or their combination for comparison, data analysis may require from on....

Results

Results from this study have been previously published¹. Figure 1 shows results from the analysis of candidate reference microRNAs using geNorm application. Accordingly, two microRNAs, miR-622 and miR-1266, were identified as reference genes and were used to normalize miRNA values.

For the CSF study we had three groups of samples: HIV- (n=10), HIVE (n=4), and HIV+ without Encephalitis (HIV+, n=5). The two groups, HIVE and HIV+, were compar.......

Discussion

MicroRNAs are small noncoding RNAs that regulate gene expression by inhibiting translation and/or promoting mRNA degradation². Due to their high stability in cell-free conditions, microRNAs have been detected in many body fluids. Furthermore, distinct expression profile of microRNAs has been correlated with stage and/or progression in a variety of human cancers^3-9.

There are several tools available to profile microRNAs, including classical chip arrays or the latest deep s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Table 3. List of equipment.
Finnpipette Novus Multichannel Pipetter	Thermo Scientific	HH05279
Finntip Pipette Tips	Thermo Scientific	9400613
Thermal Cycler C1000	Biorad
0.2 ml Low Profile, Clear PCR tubes	Biorad	TLS0801
Flat Cap Strips	Biorad	TLS0803
LightCycler 480 Real-Time PCR System	Roche
LightCycler 480 Sealing Foil	Roche	04 729 757 001
Refrigerated Centrifuge 5804R	Eppendorf
Swing-bucket Rotor, 4-place	Eppendorf	A-4-44
Bench-Top Mini Centrifuge	Fisher	05-090-100
Bench-Top Vortex	Fisher
1.5 ml Microcentrifuge Tubes, Sterilized	Fisher	02-681-5
Matrix Reagent Reservoir, 25 ml	Thermo Scientific	8093
Thermomixer Confort	Eppendorf
Bench-Top Mini Centrifuge Sigma 1-15	Sigma
Bench-Top Vortex	Velp Scientifica	F202A0173
Table 4. List of reagents.
Universal c-DNA synthesis kit, 16-32 rxns	Exiqon	203300
SYBR Green master mix, Universal RT, 25 ml	Exiqon	203400
Ultrapure Distilled Water Dnase, Rnase free	Invitrogen	10977-015
MicroRNA Ready to use PCR Human Panels (I+II) V2.R	Exiqon	203608
mirVana miRNA isolation Kit, 40 isolations	Ambion	AM1556
MS2 RNA carrier	Roche Applied Science	10165948001
2-mercaptoethanol 98%	Sigma Aldrich	M3148-25ml
Ethanol 100%	Carlo Erba	64-17-5
Nuclease free water	Qiagen	1039480
RNAse Zap	Ambion	AM9780