MicroRNA PCR Services  We now offer the most accurate, sensitive and robust PCR system for microRNA analysis available: miRCURY LNA™ Universal RT microRNA PCR. Whether you wish to perform genome-wide profiling or analyze a limited number of microRNAs, we provide access to expert scientists and state-of-the-art laboratories for maximum reproducibility. We help you identify the optimal combination of endogenous controls and calculate relative quantification, based on the amplification efficiency-corrected ΔΔCq method to ensure that you receive accurate results. Our data quality assessment, including melting curve- and negative control analysis, ensures that all data derives from specific amplification products. Our PCR Services covers everything from initial consultation to the final report.
Niels M. Frandsen, Ph.D., Product Manager | |
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When you place an order with Exiqon's microRNA PCR Services, you are assured direct communication with the scientists performing your experiments throughout the duration of the project. Each service project begins with a free consultation with an experienced PCR specialist. Together we try to identify the experimental setup and data analysis that best suits the objectives of your project. We also offer crucial advice on how to obtain high quality RNA samples. |
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Figure 1
Workflow of a microRNA PCR Services project (Click to learn more) |
Before sending us samples we kindly ask you to complete a sample submission form (see link to the right). Technical specialists will review the information to make sure that there are no outstanding questions and will then ask you to ship your samples. Please find some guidelines on sample preparation, size and type below.
Sample preparation High quality sample preparation is important for accurate microRNA quantification. First, it is crucial to use extraction methods that allow purification of total RNA which includes small RNAs. Second, it is important to avoid degradation of the RNA. Third, the purity of the preparation is very important for high quality data. Certain types of samples, such as FFPE, serum and plasma require special extraction protocols. At the initial consultation, we will offer recommendations on suitable extraction and clean-up methods as well as RNA quality cut-off criteria. You can also simply take advantage of our expertise and submit your samples to our RNA isolation service. It is important to keep in mind that with every manipulation of your RNA sample there is a risk of introducing an experimental bias. Therefore, we generally recommend working with total RNA rather than RNA enriched for small RNAs.
Sample size Due to the universal RT reaction and the sensitivity of our LNA™-enhanced PCR primers we can perform many different assays on the same sample with minute RNA input. We normally use 30ng for cDNA synthesis (triplicate RT reactions). However, if needed, we can perform analysis with much less RNA. Customers with very small amounts of RNA should consult us for further advice, since our assays are sensitive down to the low pg range. An additional 100ng is used for RNA quality control.
Sample typeWe work with total RNA from all sample types such as cell line cultures, whole blood, serum, plasma and other bodily fluids, animal tissue and clinical samples. For submissions of enriched RNA samples we request that you, in addition to the enriched sample, submit at least 100ng of the corresponding total RNA for quality control and quantification purposes.
RNA quality control After receiving your RNA samples, our specialists will assess the integrity of each sample and determine its concentration spectrophotometrically using Bioanalyzer and NanoDrop™ instruments. The results of this assessment will be made available to you on Exiqon’s secure web server. |
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Figure 2
Accurate quantitation from down to 1 pg total RNA starting material. (Click to learn more)
Figure 3
Detection of of microRNAs in LCM specimens. (Click to learn more) |
Our state-of-the-art laboratories use high throughput robotic pipetting stations that ensure a level of reproducibility not easily obtained by manual pipetting. Master mixes are setup on a Beckman Coulter Biomek® 3000 robotic pipetting station in a template-free environment to minimize the risk of contamination. Templates are handled in a separate location on an Eppendorf epMotion™ 5075 LH automatic pipetting robot. Quantitative real-time PCR is performed on a Roche LightCycler™ 480 using SYBR® green-based detection.
Technical replicatesWe recommend three RT reactions per sample and one real time PCR reaction per cDNA. This is because the RT reaction contributes more to variation of the real-time PCR analysis than the PCR amplification itself. This set-up will result in three data points per RNA sample and assay.
ControlsWe perform three negative controls per assay: - A no RNA control to check for nucleic acid contamination and primer dimer formation.
- A no reverse transcriptase control to check for contamination from genomic DNA.
- A no PCR template control to check for nucleic acid contamination of PCR reagents and problems with PCR primer dimer formation.
Positive controlAs a positive control for each assay used we perform RT reactions and subsequent real-time PCR analysis on complex universal reference RNA made from pools of Ambion first choice® total RNA.
Endogenous controlsAccurate normalization of PCR data requires identification of endogenous controls that are expressed at constant levels in your samples. It is very difficult to predict which controls are appropriate for a given experiment; they have to be determined experimentally. We have developed a panel of endogenous control assays based on small stable RNAs as well as generally stably expressed microRNAs. For standard projects we recommend using five endogenous controls. If you have prior knowledge about your samples from microRNA profiling with either miRCURY LNA™ arrays or our ready-to-use PCR human panels we might be able to narrow down the number of endogenous controls required for your project. |
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Figure 4
Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System. (Click to learn more)
Figure 5
Excellent day-to-day reproducibility. (Click to learn more)
Figure 6
Expression levels of reference genes in different human tissues. (Click to learn more) |
Before proceeding with the data analysis, we analyze the quality of the data in several ways:
The use of SYBR® green dye allows us to analyze each PCR product by generating melting curves. More than one peak will alert us to potential problems of unspecific amplification products that may confound the analysis. It is worth noting that this kind of analysis is unique to dye-based PCR systems and cannot be performed with probe-based systems.
In addition, we evaluate the Cp values of the negative and positive controls and perform a technical replicate variation assessment.
Finally we check that the amplification efficiency of each individual PCR amplification is within our accepted range: 1.8<E<2.15 |
Figure 7
Specific and sensitive amplification of microRNA. (Click to learn more)
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Real-time PCR is one of the most sensitive and accurate ways of quantifying microRNAs. However the outcome of real-time PCR assays is highly sensitive to small variations in sample input, sample purity and cDNA synthesis. Two common sources of gross misinterpretation of PCR data is 1) inaccurate normalization and 2) failure to correct for variations in PCR amplification efficiency.
NormalizationIn order to identify the relevant biological differences in your samples, it is important to normalize your data using endogenous control RNAs that are expressed at constant levels in your samples. Often expression of a single endogenous control is not sufficiently stable for accurate normalization. Choosing the right endogenous control or combination of controls is a delicate part of real-time PCR analysis. Based on the results with the endogenous control assays, we use the advanced SLqPCR R package (similar to geNorm) to select the most appropriate combination of endogenous controls for your study. A normalization factor for each sample is then calculated based on the geometric mean of the selected endogenous controls.
Measurement of PCR amplification efficiencyIn theory, the efficiency of a PCR assay should be two, since the amount of product in principle doubles for each cycle. However, for numerous reasons amplification efficiency is not always constant and not always 2. This can be a property intrinsic to the PCR assay but it is often the result of the presence of PCR inhibitors or enhancers in your samples. We determine the efficiency of each individual PCR amplification in your project with the LinRegPCR quantitative PCR analysis program (v.11.0).
Relative quantificationWe perform relative quantification using the amplification efficiency corrected ΔΔCq method. Since the amplification efficiency is not always constant and not always two, a correction for efficiency differences between samples and between microRNA target and endogenous control assays must be made. Without amplification efficiency correction, i.e., using the standard ΔΔCq method, it is risky to compare data from different sample groups or different assays and to compare with data from other platforms such as microarrays. If amplification efficiencies vary between sample groups or assays, gross errors of interpretation may occur.
Absolute quantificationWe offer absolute quantification based on external calibration curves generated with 8 dilutions of synthetic oligo corresponding to your microRNA target.
Additional data analysisWe also perform statistical and other data analysis appropriate for the experiment at hand, such as T-test or ANOVA, unsupervised or supervised clustering illustrated with two dimensional heat maps, PCA plots and analysis of correlation with microarray data.
Final reportWhen the service order has been completed you will receive an e-mail with a link to our secure web-server from which the final report and all associated files can be downloaded. A summary report is provided containing a description of the project and an overview of the results and an assessment of the quality of the data. An extensive Excel file will contain raw Cp values, melting curves and amplification efficiencies for each PCR product, negative and positive control data and analysis of variation between replicates. Stability scores of the endogenous controls are calculated and a normalization factor is calculated based on the best combination of controls. Finally relative quantification is expressed as amplification efficiency corrected ΔΔCp ratios. In addition, we report the results of various types of statistical and other types of data analyses performed on your project along with publication grade illustrations. |
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Figure 8
MicroRNA qPCR and microarray profiling compared. (Click to learn more) |
MicroRNA profiling with our ready-to-use PCR human panels is a powerful alternative to microarray profiling – especially for applications where the RNA amount available is very limited, such as precious clinical serum/plasma samples (or other bodily fluids) or FACS sorted cells. You need not worry about bias introducing steps involving complex mixtures of RT primers for cDNA synthesis and PCR primers for pre-amplification. Due to the simplicity of the universal reverse transcriptase reaction and the unique sensitivity and selectivity of ourLNA™-enhanced PCR primes we can offer you global microRNA PCR profiling using very small amounts of RNA. We perform high quality profiling of 731 human microRNAs with detection of even low abundant species using only 40ng of total RNA or RNA corresponding to just 65µl of serum. The PCR panels are ideal for discovery projects and pre-screenings on a limited number of samples to identify the microRNAs that vary in your system and carry information. Subsequently this subset of microRNAs can then be analyzed cost effectively on a much larger amount of samples. PCR panels are also relevant with large complex projects where many different questions are asked – for example when the task is to correlate microRNA expression to a wide variety of clinical parameters. |
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Figure 9
The benefit of a universal RT step. (Click to learn more)
Figure 10
MicroRNA profiling in serum and FFPE samples. (Click to learn more) |
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