MicroRNAs (miRNAs) are small, noncoding RNAs approximately 22 nucleotides in length. Highly abundant in both animals and plants, they regulate the expression of thousands of genes in both normal and pathophysiological processes and their aberrant expression is associated with many types of cancer and other diseases.
Numerous studies have shown that miRNAs have great potential as biomarkers for a wide range of medical and veterinary applications including diagnostics, prognostics, nucleic-acid-based therapeutics and theranostics. Investigators can monitor miRNA in samples from tissues, cells and body fluids such as urine, cerebrospinal fluid, blood, plasma and serum.
The most accurate and specific approach to miRNA profiling is quantitative real-time PCR (qPCR). The technique requires only minimal sample and has a broad dynamic range, accurately quantifying miRNA targets ranging from 10 to 107 copies in a single assay. With qPCR an entire miRNome representing more than 2,500 human miRNAs can be profiled using just 1.5 µg of total RNA.
Sometimes, though, that amount of RNA can be hard to come by, yet the desire to profile many miRNAs or even the miRNome remains. Here we offer tips for overcoming small sample limitations and special considerations for utilizing different types of limiting samples.
Integrate preamplification
MicroRNA profiling involves RNA purification, cDNA synthesis and qPCR. With preamplification, the entire miRNome can be profiled from samples containing as little as 10 ng of total RNA. Preamplification kits such as QIAGEN’s miScript™ PreAMP PCR Kit and Primer Mixes use a PCR approach to amplify the cDNA without bias, producing sufficient material to run plates of miRNA qPCR assays or even profile the entire miRNome. Incorporating preamplification strategies into experiments starting with blood, urine and other biofluids can increase the cDNA concentration 1,000-fold or more, effectively allowing researchers to start with less to make the most from their precious samples.
Efficiency and control
Incorporating miRNA isolation controls into your miRNA analysis can help you monitor the reproducibility and quantity of RNA isolated from each sample. Adding additional controls to monitor the reverse transcription and qPCR steps can add additional validity to the data set and provide evidence that the enzymatic steps necessary for qPCR are working optimally.
The RNA purification and reverse transcription control is usually a single-stranded non-primate RNA (such as QIAGEN’s synthetic version of the Caenorhabditis elegans miRNA, cel-miR-39-3p), which is spiked into the sample before RNA purification, while the reverse transcription control is another single stranded synthetic RNA added to the RNA sample after isolation and prior to reverse transcription. The PCR control is usually an artificial DNA which is used to monitor for contaminants and inhibitors that could negatively impact amplification itself. When all three controls are used in tandem the technical variables of PCR can be easily monitored on a sample-by-sample basis, enabling researchers to exclude potentially invalid data.
Sample normalization controls are of particular importance for data analysis using the ΔΔCT method of relative quantification. Changes in miRNA levels associated with disease can be modest, meaning precise measurements that account for fine differences in sample input is a must. Typical normalization controls for cellular samples such as tissue biopsies are noncoding RNAs such as small nuclear and nucleolar RNAs (snRNAs and snoRNAs, respectively), but when analyzing circulating miRNA (such as in blood, serum or plasma) other methods must be used. These special considerations are discussed below.
Special Considerations
Blood samples
There are three types of blood samples: Whole blood, the nucleated cell fraction and the extracellular fraction (plasma or serum). miRNAs are surprisingly stable in extracellular fluids, where they are contained in exosomes and other microvesicles, and also occur as complexes with specific proteins. Plasma centrifuged from EDTA- or citrate-anticoagulated whole blood is preferred to serum, which is inherently variable become of differences in clotting and serum collection. Avoid blood samples that include the anticoagulant heparin, which is a powerful reverse transcriptase inhibitor that is not removed during purification.
Whole blood and nucleated cell fractions generally contain sufficient miRNA that preamplification is unnecessary. However, even 1 µL of whole blood is enough for miRNA analysis, provided preamplification strategies are used.
Plasma and serum samples are intensively studied for circulating miRNAs that can serve as noninvasive biomarkers. Twenty microliters of such samples is often sufficient for qPCR, but preamplification can yield a marked increase in detected miRNAs, especially if the miRNAs are present in low abundance. Complete miRNome profiling can often be performed after preamplification from as little as 1 µL of plasma.
Though normalization controls for cellular samples such as tissue biopsies are generally noncoding RNAs, plasma and serum require alternative strategies. Options include 1) using an external RNA (such as QIAGEN’s C. elegans cel-miR-39) as a spike-in control; 2) the mean cycle threshold (CT) of all miRNA targets expressed; 3) the mean CT of commonly expressed miRNA targets common to the samples being compared; and 4) normalization based on the original volume of sample used.
Urine samples
Urine samples show promise for biomarker discovery, especially for kidney and prostate disease. The amount of miRNA in 200 µL of cell-free urine is usually not enough for more than a few qPCR assays, and processing larger sample sizes can lead to copurification of inhibitors. For cell-free analysis, clear the urine of cells and debris by centrifugation or filtering, process 100–200 µL for RNA isolation and cDNA synthesis and incorporate preamplification before qPCR. For cellular analysis, centrifuge the sample and purify the RNA from the pellet of cells and debris.
Archived FFPE samples
FFPE samples contain a wealth of information and often a clinical history and pathology report, making them ideal for biomarker discovery. Plus, unlike longer mRNAs, miRNAs generally are unaffected by the FFPE process' formaldehyde cross-linking step. Using preamplification, a 5-µm FFPE section typically yields sufficient RNA to profile the entire miRNome. Because of variations in fixation and storage, be sure to pay close attention to the reverse transcription controls and consider utilizing RNAs that will co-fractionate with miRNA and that may be less susceptible to RNA damage such as other invariant miRNAs, snoRNAs or other small non-coding RNAs.
Other small cellular samples
For sorted cells, fine-needle aspirates, microdissected cryosections and other limiting samples, perform total RNA purification and incorporate preamplification. Several miRNA isolation kits, such as QIAGEN’s miRNeasy Micro Kit, have been specially developed to isolate high quality RNA from small amounts of samples.
Each sample is unique and presents its own challenges, especially samples where RNA abundance is a concern. In the past few years the incorporation of preamplification strategies into real-time PCR analysis has enabled researchers to squeeze maximal data from minimal amounts of sample.
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