miRNA Profiling Workflow
|miRNA expression profiling came into prominence, in part, because of the expectation that a highly expressed miRNA for a given tissue or cell type (or a developmental stage) is likely to play a regulatory role. Since the first published article to report on miRNA profiling using an oligonucleotide microarray in 2004 (Liu et al. 2004), microarray analysis has become the preferred tool for profiling miRNA expression patterns to gain insight into their relevance in development and disease. |
The NCode platform is a leading platform used to investigate miRNA expression profiling. It involves several sample preparation and microarray steps and takes approximately 8-16 hrs to complete. During sample preparation, total RNA is isolated and quantified. Amplification of the small RNA molecules may be necessary where there is insufficient starting material. Next, a direct labeling method polyadenlates the enriched small RNA molecules and ligates a capture sequence to the tailed RNA. The tagged and tailed miRNA are subsequently hybridized to the array. Bound miRNAs are detected by the hybridization of branched DNA structures containing dye molecules. The miRNA expression profile is calculated from relative signal intensity detected by a microarray scanner for each spot on the array. Data validation is accomplished using qRT-PCR.
Read more on activities performed.
- Total RNA Isolation
During sample homogenization or lysis, TRIZOL® Reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components. Addition of chloroform followed by centrifugation, separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase. After transfer of the aqueous phase, the RNA is recovered by precipitation with isopropyl alcohol. > Related products.
If necessary, amplify the enriched miRNA using t is added to the miRNA using poly A polymerase and an optimized reaction buffer. Then tailed miRNA is reverse-transcribed using SuperScript™ III RT, and purified and concentrated the resulting first-strand cDNA.
Next, poly(dT) tail is added to the 3´ end of the first-strand product using terminal deoxynucleotidyl transferase, and synthesize and anneal a T7 promoter on the tailed cDNA using Klenow enzyme and a specially-designed T7 template oligo. Finally, an in vitro transcription reaction is performed with an overnight incubation to generate the amplified senseRNA. > Related products.
- Labeling and Hybridization
The NCode™ miRNA Rapid Labeling System tags the miRNAs in a simple 1-hour procedure and hybridizes them to the microarray. Using the Labeling System, a poly(A) tail is added to the miRNA using poly A polymerase and an optimized reaction buffer (this step if unnecessary if you are using amplified miRNA). Then a short, highly specific tag sequence is ligated to each tailed miRNA using a bridging oligo. Following a purification step, the tagged miRNA is hybridized to the microarray and incubated . Next the array is washed , hybridized with Alexa FluorR 3 and Alexa FluorR 5 capture reagents, washed again, and scaned using a standard microarray scanner. The capture reagents are comprised of DNA polymers, each with ~900 Alexa FluorR molecules bound to a sequence complementary to the ligated tags on the hybridized miRNAs. The high specificity of the binding sequence and high fluorescence of the dye molecules ensure maximum signal-tobackground ratios and strong signal correlations. > Related products.
- Data Analysis
Microarray signal intensities detected by the scanner are quantified by detection software. Signal intensities for each spot are calculated by subtracting local background and raw data are normalized and analyzed by using data analysis software. Learn more on data analysis methods typically used for normalization of 2-dye expression profiling microarray experiments.
- Data Validation Using qRT-PCR
Initially, the miRNAs are polyadenlyated using poly A polymerase and ATP. Following polyadenylation, SuperScript™ III RT and a specially-designed Universal RT Primer are used to synthesize cDNA from the tailed miRNA population. Subsequently, the first-strand cDNA is analyzed in qPCR using SYBR® Green or SYBR® GreenER™ detection reagents, a universal qPCR primer provided in the kit, and a forward primer designed by the user that targets the specific miRNA sequence of interest. > Related products.