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.
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.
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 statistical analysis and graphical representation of results. The entire procedure is relatively easy and straightforward and, depending on the number of samples to be profiled and the number of real time PCR machines available, also relatively quick. The experimental part requires accuracy in handling RNA and pipetting into 384-well plates, while the data analysis section using GenEx requires some basic knowledge in informatics and statistics.
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 one to several hours.
1. RNA Extraction
The RNA extraction was performed from CSF samples, stored frozen at -80 °C in aliquots until analysis. For this procedure the mirVana Paris isolation kit was used, following the manufacturer's instructions for the total RNA isolation. Please note that, although enrichment for small RNA species is possible with this kit, this step is not done and the RNA extraction procedure is ended after the total RNA isolation step. In addition, although the mirVana Paris isolation kit (like other commercially available RNA isolation kits) does not require organic extraction, a protocol for this procedure can be found below.
The protocol for RNA extraction consists of two steps:
1.1. Organic Extraction (not required if using mirVana Paris RNA extraction kit)
1.2.2. Total RNA isolation
1.1. Organic extraction
1.1.1. Prepare reagents
1.1.2. Prepare the CSF
1.2. RNA isolation
It is recommended to have a dedicated bench, set of pipettes, and racks for handling RNA samples. The working area and tools are decontaminated by using RNase Zap spray and wipes prior each experiment.
1.2.1. Prepare reagents:
1.2.2. Total RNA isolation
2. miRNA Profile: qRT-PCR Protocol
The protocol for miRNA profiling consists of two steps:
2.1. First-strand cDNA synthesis
2.1.1. Dilute template RNA
2.1.2. Prepare reagents
2.1.3. Reverse transcription reaction setup
Reagent | Panel I Volume (µl) | Panel I + II Volume (µl) |
5x reaction buffer | 4.4 | 8.8 |
Nuclease free water | 9.9 | 19.8 |
Enzyme mix | 2.2 | 4.4 |
UniSp6 RNA Spike-in template | 1.1 | 2.2 |
Template total RNA (5 ng/µl) | 4.4 | 8.8 |
Total Volume | 22 | 44 |
2.1.4. Mix and spin reagents
2.1.5. Incubate and heat inactivate
2.2. Real-time PCR amplification
2.2.1. Prepare reagents for Real-time PCR
2.2.2. Combine SYBR Green master mix, water, and cDNA and add to PCR plates
The following procedure is recommended to avoid low concentrations of cDNA from adhering to tube surface:
2.2.3. Real-Time PCR
Perform real-time PCR amplification and melting curve analysis following the parameters detailed in Table 2.
Process Step | Settings, Roche LC480 |
Polymerase Activation/Denaturation | 95 °C, 10 min |
Amplification | 45 Amplification cycles at 95 °C, 10 sec 60 °C, 1 min Ramp-rate 1.6 °C/sec Optical read |
Melting curve analysis | Yes |
Table 2. Real-time PCR cycle conditions using the Roche LightCycler 480.
2.3. Real-time PCR data analysis
Real time PCR data analysis is done with the Exiqon GenEx Professional 5.0, following the recommended instructions. The GenEx step-by-step manual can be downloaded at http://www.exiqon.com/ls/Documents/Scientific/Exiqon-data-analysis-guide.pdf
Data analysis consists of three steps:
2.3.1. Import of data
2.3.2. Preprocessing of data
2.3.3. Statistical Analysis
GenEx software allows a wide range of statistical analyses, including T-test and ANOVA.
2.3.4. Expression profiling
MicroRNAs and samples can be classified, clustered, and visualized on heat-maps and dendrograms, as follows
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 compared with the control HIV- group. After statistical analysis (section 2.3.3) expression levels of eleven microRNAs was found statistically significant1. Figure 2 represents a box plot of this eleven microRNAs, miR-1203, -1224-3p, -182*, -19b-2*, -204, -362-5p, -484, -720, -744*, -934, and -937. Each column shows the distribution of data across the samples within the group (green for the HIVE and red for HIV+ without encephalitis). Whiskers indicate the median, the 1st (Q1) and 3rd (Q3) quartile, and the maximum and minimum nonoutliers.
Figure 1. Graphic bar showing results from geNorm application within GenEx. Ten microRNAs were analyzed as possible reference genes and results indicate miR-622 and miR-1266 (red bars) as the most stably expressed miRNAs. Click here to view larger image.
Figure 2. Box plot showing the distribution of data for each of the eleven miRNAs within the groups of HIVE (green) and HIV+ without encephalitis (HIV+, red). The whiskers in each column indicate the median, 1st (Q1, bottom of the box) and 3rd (Q3, top of the box) quartiles, maximum and minimum values that are nonoutliers (black lines). Click here to view larger image.
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 cancers3-9.
There are several tools available to profile microRNAs, including classical chip arrays or the latest deep sequencing technology. However, we opted to utilize a highly sensitive qPCR platform10,11, which has the additional advantage of requiring minimal amount of RNAs. The miRCURY LNA Universal RT microRNA PCR protocol is optimized to use 20 ng total RNA per 20 µl cDNA synthesis reaction, 40 ng for the full two-panel array. Having the option of using small amount of RNA is highly important when dealing with valuable and hard-to-obtain clinical specimens such as CSF or formalin-fixed paraffin-embedded tissues1. Importantly, utilizing low amounts of RNA minimize the concentration of possible inhibitors present in the sample. Out of range Ct values for the spike-in will likely indicate the presence of some inhibitors in the sample. Spike-in probes can be purchased separately to test the samples for the presence of inhibitors prior to miRNA profiling. For this test, single real time PCR are performed using increasing concentrations of RNA (whether ng or µl).
In the present protocol, we report the organic phase extraction prior to RNA isolation. It should be noted that many new RNA extraction kits do not require this procedure. For instance, we have successfully profiled plasma miRNAs using the miRCURY RNA isolation kit biofluids for RNA extraction directly from 200 µl of plasma (data not shown), without phenol/chloroform extraction.
In general, it is important to design the experiment in advance in order to determine the proper number of replicates needed to obtain statistically significant results. The number of replicates may depend on the number of samples to be analyzed and may depend on the variations within the samples or group of samples. For our study, for which we used a relatively low number of samples, 20 in total, we decided to set up the cDNA synthesis reaction in triplicates. Consulting with a biostatistician before setting up the experiments may be a wise choice and is highly recommended.
Defining the Ct cut off value is important and depends on the type of samples; for highly expressed miRNAs it can be set between 25-35, but for low expressed miRNAs, such as our CSF specimens, can be set higher. Another critical step when it comes to data analysis is the choice of reference gene(s). For some robust data (such as miRNA profiling in cultured cells) a global normalization, which represents the average Ct of all samples, may be used. However, this is not an option for samples like CSF that present variations. Similarly, we couldn't utilize as reference genes those miRNAs that are generally considered unchanged in body fluids. Instead, we selected all miRNAs that showed similar Ct across all samples, including replicas, and ran the geNorm analysis available in GenEx (Figure 1). Results indicated miR-1266 and miR-622 as the least variably expressed miRNAs and they were used as reference genes. Comparison of the expression of miRNAs between groups can be determined using the relative quantification tool in GenEx, which follows the standard formula 2-(Ct-Ctrel). Finally, results can be visualized as heat map diagrams, graphic bars or other types of plots, such as the box plot in Figure 2. Other types of visualization available in GenEx include hierarchical clustering, Self-Organized Map (SOM), and Principal Component Analysis (PCA). In addition to miRNAs, the GenEx software can be used for the analysis of other types of arrays such as long noncoding RNAs (lncRNAs) profiling. The plate layout can be imported in the excel format and data can be handled as described for miRNA analysis. Indeed, we have profiled lncRNAs from primary embryonic mouse cortical neurons using the mirVana miRNA isolation kit as described in the steps above and we analyzed data using GenEx (unpublished results).
In summary, we described a protocol to profile microRNAs in the cerebrospinal fluid. With some modifications related to RNA extraction, this procedure can be adapted to profile miRNAs in other body fluids and/or other type of tissues. Note that in the procedure described here, a step of organic extraction is performed prior to RNA isolation. In general, this is not required when using commercially available kits, such as the miRVANA Paris extraction kit. In addition, when extracting RNA from body fluids and a carrier RNA is added to the samples there is no need to quantify the RNA and 2-8 µl RNA are subjected to cDNA synthesis. From our experience and considering the high costs related to this type of experiments, we recommend testing few samples first, and then consulting with a statistician for the number of samples/replicas to be profiled in order to obtain statistically significant results.
The authors have nothing to disclose.
The project described was supported by Award Number R01MH079751 (PI: F. Peruzzi) from the National Institute of Mental Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Mental Health or the National Institute of Health.
Table III. List of equipment
Equipment |
Company |
Catalog Number |
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-To Mini Centrifuge Sigma 1-15 |
Sigma |
|
Bench-Top Vortex |
Velp Scientifica |
F202A0173 |
Table IV. List of reagents
Reagents |
Company |
Catalog Number |
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 |
2Betamercaptoethanol 98% |
Sigma Aldrich |
M3148-25ml |
Ethanol 100% |
Carlo Erba |
64-17-5 |
Nuclease free water |
Qiagen |
1039480 |
RNAse Zap® |
Ambion |
AM9780 |