MystiCq^® MicroRNA^® Quantitation System

Technology Overview
Workflow
Background

Protocols
miRNA isolation
cDNA synthesis
miRNA qPCR

Advanced information
Product selection guides
Troubleshooting

Workflow

Figure 1. microRNA Workflow

Background

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

Protocols

microRNA isolation using RNAzol^® RT

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.

Reagents

• RNAzol^®RT (R4533)
• 75% Ethanol
  • 200 proof absolute), for molecular biology (E7023)
  • Water for molecular biology (W4502)
• 100% isopropanol (I9516)

Procedure

A. Sample Preparation- Select appropriate sample type:

1. Tissue: Homogenize tissue samples in RNAzol^® RT (1 mL per up to 100 mg of tissue) using a Polytron^® or other appropriate homogenizer.
   Note: If the tissue has high DNA content (e.g. spleen tissue), use 1mL of reagent per 50 mg of tissue.
2. Monolayer cells: Lyse cells directly on the culture dish. After removing culture medium, use at least 1 mL of RNAzol^® RT per 10 cm² of glass culture plate surface area. After addition of the reagent, the cell ysate should be passed several times through a pipette to form a homogenous lysate.
3. Suspension cells: Isolate cells by centrifugation and then lyse in TRI Reagent by repeated pipetting. 1 mL of the reagent is sufficient to lyse 5–10X10⁶animal, plant, or yeast cells, or 10⁷bacterial cells. Notes:
   i. Some yeast and bacterial cells may require a homogenizer.
   ii. After the cells have been homogenized or lysed in TRI Reagent, samples can be stored at –70 °C for up to 1 month.
4. Liquid Samples: Lyse using 1 mL RNAzol^® RT per 0.4 mL of sample. For small volume samples, supplement the sample with RNase-free water to reach a sample volume of 0.4 mL prior to addition of 1 mL RNAzol® RT.
   

B. MicroRNA Isolation

1. Transfer the aqueous phase to a fresh tube. Centrifuge a second time. Transfer aqueous phase to a clean tube
2. Add 0.4 mL of RNase-free water per mL of RNAzol^® RT used for homogenization. Cover the sample tightly, shake vigorously for 15 seconds, and allow to stand for 5–15 minutes at room temperature.
3. Centrifuge the resulting mixture at 12,000 x g for 15minutes at 4-28 °C. Centrifugation separates the mixture into a semisolid pellet (containing DNA, proteins and polysaccharides) and an upper supernatant (containing RNA). Transfer supernatant to a new tube, leaving a layer of the supernatant above the DNA/protein pellet.
   Notes:
   i. Samples homogenized with 1 mL RNAzol^® RT per 100 mg tissue require a 15 minute standing time.
   ii. Up to 85% of the supernatant can be collected for isolation of RNA.
4. Transfer the supernatant to a fresh tube and add 0.4 mL of 75% ethanol (v/v) to precipitate mRNA.
5. Allow the sample to stand for 10 minutes at room temperature.
6. Centrifuge at 12,000 x g for 8 minutes at 4-28 °C. The mRNA precipitate will form a white pellet on the side and bottom of the tube.
7. Transfer the supernatant to a clean tube, being sure not to disturb the pellet.
8. Notes:
   i. This supernatent contains mRNA and other RNA >200 bases, including large nuclear RNA and ribosomal RNA.
   ii. The micro-RNA containing supernatant can be stored at –20 °C for one year.
9. Add 0.8 volumes of 100% isopropanol to the supernatent.
10. Allow sample to stand for 30 minutes at 4 °C.
11. Centrifuge at 12,000 x g for 15 minutes at 4-28 °C. The micro RNA precipitate will form a white pellet on the bottom of the tube.
    Note: This fraction contains micro RNA and other RNA <200 bases, including small ribosomal RNA and tRNA.
12. Wash microRNA pellet twice with 0.4-0.6 mL 75% ethanol (v/v) and 70% isopropanol (v/v), respectively, per 1 mL of supernatant used for precipitation. Centrifuge at 4,000-8,000 x g for 1-3minutes at room temperature. Remove alcohol solution with a micropipette.
13. Solubilize the RNA pellets, without drying, in RNase-free water at a concentration of 0.1 μg/μL. Vortex the samples at room temperature for 2-5 minutes.
14. Notes:
    i. Drying the RNA pellet can decrease solubility and is not recommended.
    ii. Final preparation of micro RNA is free of DNA and proteins. It should have a A₂₆₀/A₂₈₀ ratio of 1.6-1.7 and a A₂₆₀/A₂₃₀ ratio of about 1.5.

Troubleshooting Guide- RNA Isolation

Low RNA Yield	incomplete homogenization or lysis of samples
	the final RNA pellet may not have been completely dissolved
A260/A280 ratio is <1.65	the amount of sample used for homogenization may have been too small
	samples may not have been allowed to stand at room temperature for 5 minutes after homogenization
	there may have been contamination of the aqueous phase with the phenol phase
	the final RNA pellet may not have been completely dissolved
RNA degradation	the tissues may not have been immediately processed or frozen after removing from the animal
	the samples used for isolation or the isolated RNA preparations may have been stored at –20 °C instead of –70 °C as specified in the procedure
	cells may have been dispersed by trypsin digestion
	aqueous solutions or tubes used for procedure may not have been RNAse-free.
	formaldehyde used for the agarose gel electrophoresis may have had a pH value <3.5
DNA contamination	the volume of reagent used for the sample homogenization may have been too small
	samples used for the isolation may have contained organic solvents (ethanol, DMSO), strong buffers or alkaline solution

microRNA cDNA Synthesis using MystiCq^® microRNA cDNA Synthesis Mix

Assay Considerations

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.

Reagents

• MystiCq^® microRNA cDNA Synthesis Mix Kit (MIRRT), includes the following components:
  • Poly (A) Tailing Buffer (5X), MIRRT01
  • Human Positive Control Primer, MIRRT02
  • Nuclease-free Water, MIRRT03
  • MystiCq™ Universal PCR Primer, MIRRT04 or MIRUP
  • Poly (A) Polymerase, MIRRT05
  • MystiCq™ microRNA cDNA Reaction Mix, MIRRT06
  • ReadyScript Reverse Transcriptase, MIRRT07
• Tubes, select one of the following to match desired format:
  • Individual thin-walled 200 µL PCR tubes (Z374873 or P3114)
  • Individual thin-walled 650 µL PCR tubes (Z374873)

Procedure

Poly(A) Tailing Reaction

1. After thawing components (except enzyme), place components on ice. Mix, and then briefly centrifuge to collect contents at the bottom of the tube.
2. Combine the following reagents in 0.2-mL micro-tubes or a 96-well plate sitting on ice.

Reagent	Volume
Poly (A) Tailing Buffer (5X)	2 μL
RNA (up to 1 μg total RNA or microRNA-enriched RNA)	Up to 7 μL
Nuclease-free water	variable
Poly (A) Polymerase	1 μL
Total Volume	10 μL

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:

• 60 minutes at 37 °C
• 5 minutes at 70 °C

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:

Reagent	Volume
Poly (A) Tailing Reaction (from final step above)	10 μL
MystiCq® microRNA cDNA Reaction Mix	9.0 μL
ReadyScript Reverse Transcriptase	1.0 μL
Total Volume	20 μL

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.

• 20 minutes at 42 °C*
• 5 minutes at 8.5 °C

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.

Real Time SYBR Green RT-qPCR Amplification of MicroRNAs

Assay Considerations

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

• The design of highly specific primers is the single most important parameter for successful real-time PCR with SYBR Green I dye.
• Predesigned primers that meet stringent design criteria are available

Product Description	Product Number Range
MystiCq® microRNA qPCR assay primer	MIRAP00001 through MIRAP01434
MystiCq® microRNA qPCR control primer	MIRCP00001 through MIRCP00016
MystiCq® Universal PCR primer	MIRUP

• The use of computer aided primer design programs is encouraged in order to minimize the potential for internal secondary structure and complementation at 3’-ends within each primer and the primer pair.

Amplicon size

• MystiCq^® microRNA^® SYBR^® Green qPCR ReadyMix™ can readily amplify fragments between 400 and 500 bp; however, for best results, amplicon size should be limited to 80 - 200 bp.
• Optimal results may require titration of primer concentration between 100 and 500 nM. A final concentration of 300 nM for each primer is effective for most reactions.

Assay setup

• Preparation of a reaction cocktail is recommended to reduce pipetting errors and maximize assay precision.
• Assemble the reaction cocktail with all required components except sample template (genomic DNA or cDNA) and dispense equal aliquots into each reaction tube.
• Add the DNA template to each reaction as the final step. Addition of samples as 5 to 10-µL volumes will improve assay precision.

Template concentration—Suggested input quantities of template are:

• cDNA- corresponding to 1 pg to 100 ng of total RNA
• genomic DNA- 100 pg to 100 ng genomic DNA

Reagents

Primer

Name	Product Numbers	Description
MystiCq® microRNA qPCR Assay Primer	MIRAP00001 through MIRAP01434	Experimental microRNA sequences designed for human, mouse and rat (may cross-react with other species)
MystiCq® microRNA qPCR Control Primer	MIRCP00001 through MIRCP00016	Control primers
MystiCq® Universal PCR Primer	MIRUP	Complementary to adapter sequence added during cDNA synthesis
Visit our MystiCq® product page for an up to date product list

Select qPCR reagent based upon instrument used:

	MystiCq®µRNA® SYBR®qPCR ReadyMix™	MystiCq®µRNA® SYBR qPCR ReadyMix Low ROX™	MystiCq®µRNA® SYBR qPCR ReadyMix with ROX	MystiCq® µRNA® SYBR qPCR ReadyMix iQ
	MIRRM00	MIRRM01	MIRRM02	MIRRM03
Real-time PCR Instrument Name
Applied Biosystems 5700			•
Applied Biosystems 7000			•
Applied Biosystems 7300			•
Applied Biosystems 7500		•
Applied Biosystems 7500 Fast		•
Applied Biosystems 7700			•
Applied Biosystems 7900			•
Applied Biosystems 7900 HT Fast			•
Applied Biosystems 7900HT			•
Applied Biosystems StepOnePlus™			•
Applied Biosystems StepOne™			•
Applied Biosystems ViiA 7		•
Bio-Rad CFX384™	•
Bio-Rad CFX96™	•
BioRad iCycler iQ™				•
BioRad iQ™5				•
Bio-Rad MiniOpticon™	•
BioRad MyiQ™				•
Bio-Rad/MJ Chromo4™	•
Bio-Rad/MJ Opticon 2	•
Bio-Rad/MJ Opticon®	•
Cepheid SmartCycler®	•
Eppendorf Mastercycler® ep realplex	•
Eppendorf Mastercycler® ep realplex2 s	•
Illumina Eco qPCR	•
Qiagen/Corbett Rotor-Gene® 3000	•
Qiagen/Corbett Rotor-Gene® 6000	•
Qiagen/Corbett Rotor-Gene® Q	•
Roche LightCycler™ 480	•
Stratagene Mx3000P®		•
Stratagene Mx3005P™		•
Stratagene Mx4000™		•

PCR tubes, select tubes to match desired format:

• Individual thin-walled 200 µL PCR tubes (Z374873 or P3114)
• Plates
  • 96 well plates (Z374903)
  • 384 well plates (Z374911)
• Plate seals
  • ThermalSeal RTS™ Sealing Films (Z734438)
  • ThermalSeal RT2RR™ film (Z722553)

Protocol

• Prepare enough master mix to run all samples in duplicate.
  • Be sure to include duplicate no template negative controls (NTC).
  • Calculate amount of reagents to mix. Add 10% volume to allow for pipetting error.
• Add 25uL of master mix to each tube.

Reaction Mix:

Reagent	Volume
MystiCq® microRNA SYBR Green qPCR ReadyMix (2X)	25 μL
MystiCq® microRNA qPCR Assay Primer (10 μM) or MystiCq®microRNA qPCR Control Primer	1.0 μL
MystiCq® Universal PCR Primer (10 μM)	1.0 μL
microRNA cDNA (0.1 – 10 ng)	variable
Nuclease-free water	variable
Total Volume	50 μL

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.

• After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube.
• Cycle reactions using 2 step or 3 step PCR parameters as listed.

2-Step Cycling Procedure (recommended)

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)

3-Step Cycling Procedure (optional)

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.