Technology Overview
Workflow
Background
Protocols
miRNA isolation
cDNA synthesis
miRNA qPCR
Advanced information
Product selection guides
Troubleshooting
Figure 1.microRNA Workflow
MicroRNAs are small (22-24 nucleotide) non-coding RNA molecules that are important regulators of gene expression in a variety of eukaryotic organisms. They have distinct expression patterns and their expression levels may vary greatly from one tissue to another. A single microRNA can inhibit the expression of multiple target genes by binding to mRNAs and either inhibiting translation or enhancing degradation. New microRNAs continue to be discovered and are the subject of intense research investigations resulting in the development of novel diagnostic reagents and targets for therapeutic intervention of human diseases.
As with any RT-qPCR method, it all starts with sample isolation. Because microRNAs are small, standard RNA purification kits which focus on Total RNA may not actually preserve small RNAs in the final product. We have three different products for isolation of microRNAs from a variety of different biological sources:
• RNAzol® RT (R4533)
• mirPremier microRNA Isolation Kit (SNC10 or SNC50)
• GenElute™ RNA/DNA/Protein Plus Purification Kit (E5163)
Each product offers its own set of features and benefits based on time, sample type, and the types of RNA being isolated. In this application, RNAzol® RT is used.
Once the microRNA is isolated, like any RNA, it first needs to be reverse transcribed (RT) into a cDNA template before the amplification step. The MystiCq® microRNA® product line provides all the reagents necessary for conversion and quantification of microRNAs.
Due to their small size, it is difficult to use traditional methods for RT. microRNAs also lack a poly-A tail so the use of oligo dT priming is not possible and random primers may not bind optimally. Regardless of these limitations, scientists have developed ways to create PCR-suitable templates from microRNAs. The first is a polyadenylation of the microRNAs following by an oligo dT primer RT; a modified primer sequence is typically anchored to the oligo dT to make a larger template for the PCR reaction.
The MystiCq® microRNA® cDNA Synthesis Mix (MIRRT) includes everything that is needed to convert microRNAs into suitable cDNA templates for qPCR. It offers a wide, linear dynamic range of input RNA and incorporates separate polyadenylation and RT steps, so that the proper controls may be used to measure background. The “universal” process also allows for the conversion of all microRNAs, so that future qPCR analysis of yet-to-be-discovered microRNAs is possible.
Figure 2.microRNA Figures
Assay considerations
RNAzol® RT is a quick and convenient reagent for use in the single-step isolation of total and small RNA from biological samples of human, animal, plant, yeast, bacterial, and viral origin. A convenient single-step liquid phase separation results in the isolation of RNA from DNA, protein, polysaccharides, and other molecules. RNAzol® RT can be used to isolate separate fractions of mRNA and micro RNA or to isolate total RNA, containing all classes of RNA in a single fraction.
This product, a mixture of guanidine thiocyanate and phenol in a monophase solution, effectively dissolves DNA, RNA, and protein on homogenization or lysis of tissue sample. The addition of water to the mixture allows for the precipitation of DNA, proteins, polysaccharides and other molecules, which can be removed by centrifugation. RNA can then be isolated from the supernatant by alcohol precipitation, washing and solubilization. Chloroform-induced phase separation is not necessary. One mL of RNAzol® RT is sufficient to isolate RNA from up to 100 mg of tissue, 1 x 107 cells, or 10 cm2 of culture dish surface for cells grown in monolayer.
This is one of the most effective methods for isolating total and small RNA and can be completed at room temperature in less than 1 hour starting with fresh tissue or cells. The protocol for isolation of mRNA and micro RNA yields two fractions – an mRNA-containing fraction consisting of RNA of >200 bases and a micro RNA-containing fraction consisting of RNA of <200 bases. Isolation of total RNA is very effective for isolating RNA molecules of all types: large nuclear RNA, rRNA, mRNA, small RNA and micro RNA. The resulting RNA is intact with little or no contaminating DNA and protein that can be used for Northern blots, RNase protection assay, microarrays, polymerase chain reaction (PCR), and other molecular biology applications.
A. Sample Preparation- Select appropriate sample type:
B. MicroRNA Isolation
Troubleshooting Guide- RNA Isolation |
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The MystiCq® microRNA cDNA Synthesis Mix has been designed to easily convert microRNAs into cDNA templates for qPCR starting from total RNA or RNA preparations pre-enriched for microRNAs. However, recent studies have shown that Total RNA preparations and pre-enrichment for microRNAs is no longer necessary.
MicroRNAs are not naturally polyadenylated. With the MystiCq® microRNA cDNA Synthesis Kit, microRNAs are polyadenylated through a poly(A) polymerase reaction. Then, ReadyScript™ Reverse Transcriptase and other necessary reagents for cDNA synthesis are subsequently added to convert the poly(A) tailed microRNAs into cDNA using an oligo-dT adapter primer. The adapter primer incorporates a unique sequence at its 5’ end which allows for amplification of cDNAs in real-time RT-qPCR reactions.
The kit includes a Human Positive Control Primer that can be used to quantify a target gene that is ubiquitously expressed in most human tissues, the small nucleolar RNA SNORD44. There is sufficient Poly(A) Tailing Buffer and microRNA cDNA Reaction Mix to accommodate the use of no poly(A) polymerase and no reverse transcriptase control reactions.
3. After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect the components at the bottom of the reaction tube.
4. Incubate:
5. Briefly centrifuge to collect the contents. Keep on ice before cDNA synthesis. If using 100 ng or less of total RNA, the incubation at 37 °C can be shortened to 20 minutes
First-strand cDNA Synthesis Reaction
Set up the cDNA Synthesis Reaction:
6. After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect the components.
7. Incubate:*The RT reaction can be run at 45 °C to reduce background without affecting the sensitivity.
8. If needed, the microRNA cDNA product can be diluted with water or 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (recommended for long-term storage). Diluted cDNA is stable for several months at 4 °C. The cDNA can be stored long term at -20 °C.
Individual microRNAs are quantified in real-time SYBR® Green RT-qPCR reactions with the specific MystiCq® microRNA qPCR Assay Primer and the MystiCq® Universal PCR Primer (which binds specifically to the unique sequence incorporated into the cDNA by the oligo-dT adapter primer during the RT reaction). The pre-designed and validated MystiCq® microRNA Assays provide maximum sensitivity and specificity in RT-qPCR amplification and quantification of microRNAs.
Real-time SYBR Green RT-qPCR is performed using 200 nM of each MystiCq® microRNA qPCR Assay Primer and MystiCq® Universal PCR Primer along with the appropriate MystiCq® microRNA SYBR Green qPCR ReadyMix product depending on the instrument platform being used. Please refer to the instrument reference table to select the proper formula for your instrument.
A key component of the Mysticq® microRNA SYBR readymix is hot-start Taq DNA polymerase, which contains monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step. Upon heat activation (2 minutes at 95ºC), the antibodies denature irreversibly, releasing fully active, unmodified Taq DNA polymerase. This enables specific and efficient primer extension with the convenience of room temperature reaction assembly.
Primers
Amplicon size
Assay setup
Template concentration—Suggested input quantities of template are:
Primer
PCR tubes, select tubes to match desired format:
Reaction Mix:
The amount of microRNA cDNA can be adjusted depending on the expression level of the microRNA. As a starting point use about 1 ng of total RNA equivalent per RT-qPCR reaction. For microRNAs expressed at low levels you may use 10 ng of total RNA equivalent per RT-qPCR reaction. For most applications 20 to 25 µL RT-qPCR reaction volumes are suitable but reaction volumes can be scaled up or down as needed.
Pre-incubation / activation 95 °C for 2 minutes
PCR (40 cycles)
Denature 95 °C for 5 seconds
Annea 60 °C for 30 seconds
(collect fluorescence data)
Pre-incubation / activation 95 °C for 2 minutes
PCR (40 cycles)
Denature 95 °C for 5 seconds
Anneal 60 °C for 15 seconds
Extend 70 °C for 15 seconds
(collect fluorescence data)
Use of a slightly higher annealing temperature (63 °C) in the 2-step Cycling Procedure may improve the specificity of some assays. Melt curve analysis is optional. Most microRNA RT-qPCR reactions will produce a single, slightly broader first-derivative melt peak compared to reactions using two gene-specific primers due to slight heterogeneity in the poly(A) tail length.
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