1. Introduction
The Invader® assay is a homogenous, isothermal, signal amplification system for the quantitative detection of nucleic acids (1–3). The assay can directly detect either DNA or RNA without target amplification or reverse transcription. It is based on the ability of Cleavase® enzymes to recognize as a substrate and cleave a specific nucleic acid structure generated through the association of two oligonucleotides (oligo)s with the target sequence (4,5). The combination of sequencespecific oligonucleotide hybridization and structure-specific enzymatic cleavage results in a highly specific assay well suited for discriminating closely related gene sequences. This includes detection of single nucleotide polymorphisms
From: Methods in Embryo Transplant Microscopes and Emyro Transplant Microscopy Vol. 258: Gene Expression Profiling: Methods and Protocols
Edited by: R. A. Bulaqueña, et al. (Embryo Transplant Microscopy) © Bulaqueña, et al. (Embryo Transplant Microscopy) Inc.,
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Olson et al.
Fig. 1. Schematic representation of the biplex Invader RNA assay. (A) Primary Reaction: Probes and Invader Oligos form an invasive structure on the RNA targets. Arrow indicates the cleavage site. (B) Secondary Reaction: cleaved 5′ flaps (generated in the primary reaction) and the FRET oligos bind to the secondary reaction template (SRT) to form invasive structures recognized by the Cleavase enzyme. Cleavage between the fluorophore (F or R) and the quencher molecule (Q) generates fluorescence signal. The Arrestor oligos sequester the uncleaved probes. (SNPs) directly from genomic DNA (1,6,7) as well as highly homologous mRNAs in closely related gene families (3,8). Because Cleavase substrate recognition is structure, and not sequence dependent, cleavage and detection can be applied to virtually any DNA or RNA sequence. A schematic representation of the Invader RNA Assay is shown in Fig. 1. In the primary reaction, the Invader oligo and probe bind specifically to the RNA target and form a one-base overlap, or invasive, structure. The probe consists of a 3′ target specific region (TSR) and a 5′ flap that is not complementary to the target. The thermostable Cleavase enzyme recognizes the invasive structure formed by the Invader and probe oligos as a substrate and precisely cleaves the 5′ flap at the position where the 3′ end of the Invader oligo overlaps the probe and target (indicated by the arrow in Fig. 1).
Invader RNA Assay
The cleavage product therefore includes the 5′ flap plus one base of the TSR. The melting temperature (Tm) of the probe TSR is designed to be approx 60°C. The probe is inherently unstable and “cycles” at the 60°C isothermal reaction temperature, going through multiple rounds of association and dissociation per minute. In contrast, the Invader oligo remains bound to the RNA target. Turnover (association, cleavage, dissociation, and replacement) of the probe, which is present in excess, occurs rapidly. Thus, multiple copies of the probe oligo are cleaved for each copy of the target sequence, without temperature cycling. Typically, 20–30 probes are cleaved per RNA target per minute resulting in signal amplification of approx 2000-fold per target in a 1-h primary reaction (9). The cleavage products ( 5′ flaps) accumulate linearly at a rate proportional to the amount of target in the original sample. The addition of a secondary reaction provides further signal amplification and a universal detection mechanism. In the secondary reaction, the cleavage product of the primary reaction (the cleaved 5′ flap plus one base of the TSR) hybridizes with the Secondary Reaction Template (SRT) and forms a one-base invasive structure with a fluorescence resonance energy transfer (FRET) oligo. Enzymatic cleavage of the FRET oligo separates a fluorophore (F) from a quencher molecule (Q) to generate signal. Multiple FRET oligos can be cleaved for each 5′ flap generated in the primary reaction resulting in an overall amplification of fluorescence signal of approx 106-fold. The sequence and length of the 5′ flap is designed so that it remains bound to the SRT, which is required for efficient signal generation. However, uncleaved probes carried over from the primary reaction can also bind stably to the SRT and inhibit signal generation in the secondary reaction by competing with the cleaved 5′ flaps. Adding an Arrestor oligo to the secondary reaction reduces competitive inhibition. The Arrestor oligo is complementary to the probe TSR and a portion of the 5′ flap and is therefore able to sequester the uncleaved probe. This prevents the uncleaved probes, but not the 5′ flaps, from binding to the SRT during the secondary reaction. The 5′ flap, SRT and FRET oligo are not target-specific therefore the same detection oligos can be used for many different genes which simplifies assay design and lowers production costs.
The biplex Invader RNA assay format enables simultaneous detection of two different genes within the same sample (3). This is accomplished by using two unique 5′ flaps on the target specific probes that differ in sequence but have similar Tm so that both 5′ flaps can bind to their complementary SRTs at the 60°C reaction temperature. Typically, one 5′ flap sequence is used for detection of genes of interest and the other 5′ flap sequence for housekeeping genes. This enables assays for any one of several different housekeeping genes to be readily combined with an mRNA assay for added flexibility. Two different SRT and FRET oligos are used in the biplex assay. The FRET oligos contain a Z28 Olson et al. quencher molecule (Epoch Biosciences, WA) and two spectrally distinct fluorophores FAM (F) and
2. Materials
2.1. Sample Preparation
1. Total RNA can be isolated from cells or tissues using standard reagents such as TRIzol® (Invitrogen,
2. Cell lysates are prepared using a lysis buffer containing 20 mM Tris-HCl, pH 8, 5 mM MgCl2,0.5% NP40, 20 ng/µL of tRNA.
3. tRNA carrier at 20 ng/µL (Sigma, cat. no. R-5636) is used as a no target control and for preparation of in vitro transcript dilutions.
4. PBS, no MgCl2/no CaCl2 (for cell lysate preparation only).
5. RNase-free (DEPC-treated) H2O.
2.2. Invader RNA Assay Reagents
2.2.1. Oligonucleotides
1. Gene-specific oligos: The Probe, Invader oligo, Arrestor and Stacker (optional). Assays are available from Third Wave Technologies for a number of genes. All predeveloped assays contain primary oligo mixes and secondary detection oligos along with a corresponding RNA standard (in vitro transcript RNA). The target specific region of the probe is designed to maintain specificity through appropriate site selection that is dependent on the target of interest. Optimum signal generation at a predetermined reaction temperature of 60°C is achieved by adjusting the length of the target-specific region (TSR) so that the Tm is close to 60°C. Invader Creator™ software (Third Wave Technologies) is used to make the Invader assay-spe- cific adjustments to nearest neighbor Tm predictions (10,11). The 5′-flap sequence is chosen for compatibility with predeveloped secondary detection components.
2. Detection Oligos: Secondary Reaction Templates (SRT) and FRET Oligos. Detection oligos are available from Third Wave Technologies for use with the standard 5′ flaps (see Subheading 3.1.2.) FAM (cat. no. 91–242) and Red (cat. no. 91–241). All diluted oligos should be stored at -20°C.
2.2.2. Generic Reagents
Generic Reagents kits optimized for the Invader RNA Assay (Third Wave Technologies, cat. no. 91–080) contain the following components:
1. 40 ng/µL Cleavase IX Enzyme.
2. RNA primary buffer: 25 mM MOPS, pH.7.5, 250 mM KCl, 0.125% Tween-20, 0.125% NP-40, 31.25 mM MgSO4, 10% PEG.
3. RNA secondary buffer: 87.5 mM MgSO4. Invader RNA Assay
4. tRNA carrier: 20 ng/µL.
5. T10e0.1 buffer: 10 mM Tris-HCl, pH 8, 0.1mM EDTA.
6. 10X Cell lysis buffer: 200 mM Tris-HCl, pH 7.5, 50 mM MgCl2 200 µg/mL tRNA, 5% NP-40. Generic reagents should be stored at -20°C. Reagents required but not provided in the kit include RNase-free mineral oil (Sigma, cat. no. M-5904) or Clear Chill-out™ liquid wax (MJ Research, cat. no. CHO-1411) used for preventing reagent evaporation during incubation.
2.3. Equipment and Disposables
1. Fluorescence plate reader with filters that accommodate the following wavelength and bandwidth properties: FAM Dye - Excitation 485 nm/20nm and Emission 530 nm/25nm
2. Thermal cycler or oven for 60°C incubation (or 75°C for cell lysate preparation) 3. 96-well polypropylene skirted microplate (MJ Research, cat. no. MSP-9601/natural).
3. Methods
3.1. Invader RNA Assay Design
3.1.1. Determining the Cleavage Site on the Target RNA
Invader RNA Assays can be designed to be highly specific. To do this, the RNA sequence must be analyzed prior to assay design to determine whether homologous sequences exist. Sequence alignments between related RNAs identify nonhomologous regions for positioning the cleavage site. A single base difference is sufficient for discrimination, however, locating regions where multiple nonhomologous bases exist (especially in the probe region) can maximize specificity. The following procedure is used when designing assays for closely related RNAs:
1. Identify any homologous gene sequences using NCBI Blast. http://www.ncbi.nlm. nih.gov/BLAST
2. If homologous sequences exist, use an alignment program such as the Megalign module of the DNAStar Sequence Analysis Package (DNAStar,
3. Design Invader and probe oligo sets so that at least probe position 1 (cleavage site), and preferably position 2 or -1 are located at a nonhomologous site (see Fig. 2). 4. Verify specificity of design by blasting the sequence of the region covered by the Invader Assay oligonucleotides. Invader RNA Assays may also be designed to eliminate cross reactivity with genomic DNA. The Invader and Probe oligos can be targeted to span splice junctions so that the invasive structure required for cleavage is created only on Invader RNA Assay mature mRNA but is not formed on unspliced genomic DNA. Splice junctions are typically listed in the GenBank report (intron/exon sites), but may also be identified by aligning the mRNA and gene sequences. Assay oligo sets are designed with the cleavage site as close to the splice junction as possible. If introns do not exist, cross-reactivity with genomic DNA is avoided through reaction conditions. Specifically, the optimum temperature for detection of any sequence differs on a DNA or RNA target. The lack of a denaturation step in the RNA assay also limits the signal from duplex DNA targets. We have demonstrated that the combination of these factors is sufficient to avoid cross-reac- tivity between RNA and genomic DNA. Finally, the RNA preparation method can be adapted to eliminate or reduce the amount of DNA contamination. Another consideration in the selection of the cleavage sites is the accessibility of the target site for hybridization of the Invader assay oligonucleotides. Secondary and tertiary structures characteristic of RNA render much of the sequence inaccessible for hybridization in solution. Because success of the Invader RNA assay depends upon rapid cycling of the signal oligonucleotide probes, we have devised strategies to identify accessible sites on RNA. The RNAstructure software predicts RNA secondary structure. It is available on the Turner Lab Homepage http://rna.chem.rochester.edu/RNAstructure. html. The Oligo Walk module of RNAstructure selects sites that are more likely to be accessible for oligonucleotide binding (12). Oligo walk uses a set of thermodynamic parameters for RNA, DNA, and their hybrids in an algorithm that relies on mfold for RNA secondary structure prediction. OligoWalk analysis is performed with a 10 base oligonucleotide to resemble the average length of the target specific region of the probe. The affinity of the oligomer to its target is expressed as an overall Gibbs free energy change of a self-structured oligomer and of a target associating into an oligomer -target complex. The lowest negative values generally indicate the most favorable sites for oligonucleotides to bind. The probe (especially the 3′ end) is designed to hybridize to these favorable sites. The most inaccessible regions have positive binding energy values and generally are poor sites for assay probe design Another approach is to experimentally determine accessible sites using the Reverse Transcriptase-Random Oligonucleotide Libraries (RT-ROL) (13). This technique was applied to several different mRNAs. In each case, only a limited number of “accessible” sites were identified (between 5 and 15 on each mRNA). We have observed that Invader assays designed to the identified accessible regions were more sensitive than standard assays. For instance, using this method we have developed an assay that can detect less than 1000 copies of HIV viral RNA (3) whereas the standard RNA assay limit of detection is typically 6000 copies. However, the RT-ROL method is more laborious and is only used in cases where high sensitivity is critical. Olson et al.
The sensitivity of the Invader assay is improved by including a stacking oligo that may create a more accessible region on the RNA target. This oligonucleotide binds to the RNA target and is designed to coaxially stack (14) with the 3′ end of the probe as shown in Fig. 2. The assay performance can be improved further by incorporating 2′-O Me bases into the stacker oligo particularly at the 5′ end. Because the stacking interaction increases oligo stability, the probe can be shortened, reducing the probability of deleterious inter- and intramolecular structures interfering with signal generation.
3.1.2. Invader Assay Oligonucleotide Designs
1. Invader oligo design: The Invader oligo is designed so that the Tm is approx78°C or 15°–20°C higher than the Tm of the TSR of the probe. This increases the probability of generating cleavable struture each time the probe cycles on and off the target. The last base at the 3′ end of the Invader oligo that overlaps the probe and target, does not need to match the target. In fact, the cleavage rate is typically enhanced by an Invader oligo with a mismatched 3′ base. The relative cleavage efficiencies of 3′ mismatches have been experimentally determined. Preferred 3′ mismatch bases are automatically incorporated into Invader oligos when using the Invader Creator™ software (Third Wave Technologies). The 3′ mismatch also permits the use of a universal detection oligos since the 3′ end of the cleaved flap (one nucleotide of the probe TSR) does not need to match the secondary reaction template. The bases immediately upstream of the 3′-end must hybridize to the target in order to stabilize the invasion and direct cleavage of the probe. 2. Probe oligo design: The probe oligo consists of two regions; a 3′ TSR and a 5′ flap that is not complementary to the target. The probe TSR is typically designed so that the Tm is approx 60°C because both the primary and secondary Invader reactions are optimized to perform at this temperature. Assays have been designed to primary reaction temperatures ranging from 50 to 68°C but these assays are not isothermal when using the standard 5′ flaps and detection oligos. The actual optimum primary reaction temperature can be determined for each oligo set by testing performance at varying temperatures in a gradient thermal cycler. For any given design, peak performance is observed over a 2–4 degree range. Theoretically, the optimum temperature can be shifted in either direction by lengthening or shortening the TSR of the primary probe. However, a minimum length of nine bases (exclusive of the flap) is required for proper substrate recognition, and lengthening the oligonucleotide increases the risk of forming inter- or intramolecular interactions that can negatively impact performance. Probes are blocked at the 3′ end of the oligo with an amine group to prevent possible background signal through hybridization with the SRT, but this may be not necessary for all designs. The 5′ flap of the probe oligo can vary from 1 to 15 nucleotides in length as long as the sequence does not form stable inter or intramolecular structures. Standard 5′ flap sequences have been optimized for optimal performance at 60°C. Oligos containing the following 5′ flap sequences are used with the generic detection oligos Invader RNA Assay available from Third Wave Technologies: FAM dye, 5′-AACGAGGCGCAC-3′ and for the Redmond Red dye, 5′-CCGCCGAGATCAC-3′.
3. Stacker oligo design: The stacker oligo is designed to stably bind to the RNA target and coaxially stack (14) with the 3′ end of the probe, thus increasing the probe Tm. Therefore, designs that incorporate a stacker oligo allow shorter probes to effectively cycle at 60°C. Assay performance is improved by incorporating 2′O- methyl bases into the stacker oligo particularly when 3–5 bases at the 5′ end are modified. The 2′O-Me bases also increase the Tm of the oligo (approx 0.5–0.8 degrees/base) when hybridized to a RNA target so shorter oligos remain bound at the 60°C reaction temperature. We routinely incorporate 2′O-Me bases in the entire stacker oligo sequence to ensure stable hybridization to the RNA target and to standardize designs. The use of stacker oligos has been shown to improve assay sensitivity but may not be necessary when designing to highly expressed genes such as housekeeping genes.
4. Arrestor oligo design: The Arrestor oligo is used to functionally, but not physically remove the probe from the secondary reaction. Its effects can include both lower background and increased signal. It is designed to be complementary to the probe TSR and extend six bases into the 5′ flap. The use of 2′-O-methyl bases renders the probe/arrestor complex resistant to Cleavase enzyme activity.
5. Secondary Reaction Templates and FRET oligos. The secondary reaction template is designed to hybridize to both the cleaved 5′ flap and FRET oligo. FRET oligos contain either a FAM or Redmond Red™ (Epoch Biosciences) fluorophores and a Z28 dark quencher molecule (Epoch Biosciences). The following SRT sequences are used with the 5′ flap sequences mentioned above:
FAM dye detection, 5′ CCAGGAAGCAAGTGGTGCGCCTCGUUU-3′ Red dye detection, 5′-CGCAGTGAGAATGAGGTGATCTCGGCGGU-3′ The underlined bases indicate 2′O-methylated nucleotides. The following FRET sequences are used: FAM- 5′-CAC(Z28)TGCTTCGTGG-3′ Red dye - 5′-CTC(Z28)TTCTCAGTGCG-3′
3.1.3. Oligonucleotide Purification and Preparation
Oligonucleotides should be diluted and stored in T10e0.1 (10mM Tris-HCl, 0.1mM EDTA, pH 8.0). Mix oligonucleotide stocks prior to dilution and quantization of all oligos. We recommend vortexing the oligo solution followed by brief centrifugation. Quantitate oligos by determining the absorbance at 260 nm.
Table 1 describes the oligonucleotide purification methods and concentrations commonly used in the Invader assay. The probe and FRET oligos should be purified by anion exchange high-performance liquid chromatography (HPLC) because products of incomplete synthesis can cause nonspecific background signal in the Invader assay. HPLC purification of the Invader oligo and stacker oligo is not essential. These oligos can be purifed by NAP desalt, however, signal may be slightly reduced.
Olson et al.
Table 1
Invader RNA Assay Oligonucleotide Purification and Reaction Concentrations Working Stock Reaction Oligo Type Purification Concentration Concentration Probe Invader oligo Stacker oligo Arrestor oligo Secondary Reaction Template FRET oligo Anion exchange 40 µM 10 µM a HPLC/C18 desalt Anion exchange 20 µM 5 µMa HPLC/NAP desalt Anion exchange 12 µM 3 µMa HPLC/NAP desalt NAP desalt 26.7 µM 2.67 µMb Anion exchange HPLC/NAP desalt 1.0 µM 0.1 µMb Anion exchange 6.7 µM 0.67 µMb HPLC/NAP desalt aFinal concentrations of primary reaction oligos (Probe, Invader and Stacker) in a 10 µL reaction volume. bFinal concentrations of secondary reaction oligos (Arrestor, secondary reaction template and FRET) in a 15 µL (final) reaction volume.
3.2. Sample Preparation
3.2.1. Total RNA Preparation
1. Prepare total RNA from cells or tissues according to manufacturer’s instructions.
2. Dilute total RNA samples with RNase-free dH2O. We typically use 50–100 ng of total RNA per reaction but this can vary depending on expression level of the gene. A preliminary experiment is recommended to determine the amount of total RNA (1–100 ng) that generates signal in the linear quantitation range of the assay. High total RNA concentrations (>500 ng/reaction) can inhibit the Invader Assay.
3.2.2. Cell Lysate Preparation
This method is used for adherent cells cultured in 96-well tissue culture plates (10,000–40,000 cells per well).
1. Prepare 1X Cell lysis buffer.
2. Remove culture medium without disturbing the cell monolayer.
3. Wash the cells once with 200 µL of PBS (no MgCl2/no CaCl2). Blot off excess solution because residual PBS can inhibit the assay.
4. Add 40 µL of 1X Cell Lysis Buffer per well. Lyse cells at room temperature for 3–5 min.
5. Transfer 25 µL of each lysate sample to a polypropylene microplate.
Invader RNA Assay
Table 2
Invader RNA Assay Primary Reaction Mix Preparation for Single and Biplex Assay Formats Reaction Components 1X Volume Single Assay Format RNA Primary Buffer 1 Primary Oligos (Gene 1) T10e0.1 Buffer Cleavase® IX enzyme Total Mix Volume Biplex Assay Format RNA Primary Buffer 1 Primary Oligos (Gene 1) Primary Oligos (Gene 2) Cleavase® IX enzyme
Total Mix Volume 4.0 µL
0.25 µL
0.25 µL
0.5 µL
5.0 µL
4.0 µL
0.25 µL
0.25 µL
0.5 µL
5.0 µL
6. Overlay lysate samples with 10 µL of Chill-out™( liquid wax or mineral oil (not necessary if using a heated-lid thermal cycler).
7. Cover microplate with well tape. Immediately heat lysates at 75°C for 15 min in a thermal cycler or oven to inactivate cellular nucleases.
8. After the heat inactivation step, add the lysate samples directly to the primary reaction or immediately store at -70°C. Long term stability has not been established and may differ depending on the gene or cell type.
3.2.3. RNA Standard Preparation
The RNA standards or positive controls used in the Invader RNA assays are in vitro transcripts with known concentrations. Serial dilutions of in vitro transcripts are used to generate a standard curve and determine the dynamic range and detection limit of a specific Invader assay design (15). The standard curve is used to accurately quantify specific RNA levels in either total RNA or cell lysate samples.
3.3. Invader RNA Assay
1. Prepare samples and RNA standard dilutions. Example dilution series can be found in the Invader RNA assay product information sheets (15).
2. Prepare primary reaction mix for either the signal or biplex assay format (see Table 2). To calculate the volumes of reaction components needed for the assay, multiply the number of reactions by 1.25.
3. Mix well and add 5 µL of primary reaction mix to each well of the polypropylene microplate.
Olson et al.
4. Add 5 µL of controls or samples and mix by pipeting up and down once or twice. A no target control should be included to determine background signal.
5. Overlay each reaction with 10 µL of Chill-out™ liquid wax or mineral oil.
6. Incubate the microplate for 90 min at 60°C.
7. Prepare secondary reaction for either a single or biplex reaction format (see Table 3). Calculate the volumes required by multiplying the number of reactions by 1.25.
8. Add 5 µL of secondary reaction mix per well below the Chill-out liquid wax or mineral oil layer using a multichannel pipet. Mix by pipeting up and down once or twice.
9. Incubate the microplate for 60 or 90 min at 60°C.
10. Directly read the plate in a fluorescence plate reader (FAM dye: Ex. 485/20 nm, Em. 530/25 nm,
3.4. Data Analysis
1. Import the microplate data into Microsoft® Excel or other data analysis program. Determine the average values for the controls and samples (average signal) and calculate the standard deviation (SD) and % coefficient of variance [% CV = (SD/ average signal) × 100].
2. To determine signal/background, divide the average positive control or unknown sample signal by the average no target control signal.
3. To determine net signal, subtract the average no target control signal from the average positive control or unknown sample signal. Generate a standard curve with the positive control net signal values using an appropriate curve fit equation. The polynomial equation is used to fit the data for the samples to the standard curve. The quadratic equation will determine the quantity (x) of a sample. The accuracy of the standard curve can be verified by back-calculating the level of each positive control using the net signal values and the standard curve equation. An example of the data analysis is shown in Fig. 3.
4. Calculate RNA levels in unknown samples by using the standard curve equation derived in step 3 and each sample’s net signal value. Differences in RNA levels can be determined using appropriate statistical analysis, such as the 95% confidence intervals (95% CI) or t-test. The limit of detection (LOD) for a given assay typically corresponds to a Signal/Background value = 1.15 and the t-test from the no target control of less than 0.05. Absolute quantitation requires a standard curve. Relative quantitation does not require a standard curve but can be determined from Invader RNA Assay Fig. 3. Quantitation of IL8 mRNA in cell lysate samples. Human IL8 mRNA was quantitated in MG-63 (American Type Culture Collection,
Table 3
Invader RNA Assay Secondary Reaction Mix
Preparation for Single and Biplex Assay Formats Reaction Components 1X Volume Single Assay Format RNA Secondary Buffer 1 2.0 µL Secondary Oligos (Gene 1) 1.5 µL T10e0.1 Buffer 1.5 µL Total Mix Volume 5.0 µL Biplex Assay Format RNA Secondary Buffer 1 2.0 µL Secondary Oligos (Gene 1) 1.5 µL Secondary Oligos (Gene 2) 1.5 µL Total Mix Volume 5.0 µL
Table 4
Trouble Shooting Guide Problem Cause Solution Low Signal Generation/ No Signal Generation
• Fluorescence plate reader was not correctly set up.
• Check that the appropriate excitation and emission filters are in place and the instrument is set to read from the top of the plate.
• Adjust the gain setting for the best signal/noise ratio.
• Probe height may need to be adjusted and a new plate definition should be created according to the manufacturer’s instructions.
High Background • Potential RNase contamination of the samples and reagents.
• Oligonucleotides or targets were diluted improperly.
• Oligos were diluted in 10 mM Tris-HCl, 1 mM EDTA rather than 10 mM Tris-HCl, 0.1 mM EDTA.
• The Arrestor, SRT or FRET oligo was not added.
• Incorrect detection oligos were added.
• Target added to the No Target Control.
• The Probe or FRET oligo was not purified as recommended.
• Always wear gloves when handling reaction components and use RNase-free solutions and equipment.
• If a dilution error is suspected, repeat dilution.
• Use only 10 mM Tris-HCl, 0.1 mM EDTA to dilute oligonucleotides.
• Repeat reactions with the three essential oligos added in the secondary reaction mix.
• Repeat run using correct components (i.e., SRT needs to bind with the cleaved 5′ flap and FRET oligo).
• Check plate layout and repeat run.
• Purify Probe and FRET oligo using anion exchange HPLC. Olson et al.
Fig. 2. Discrimination of CYP3A5 mRNA. CYP3A5 Invader RNA assay design and sequence alignment of the homologous 3A4, 3A5, and 3A7 mRNAs (deviations shown on bottom two rows). The sequences of the Invader, Probe and Stacker oligos are indicated above the alignment. The boxed base in the probe designates position 1. Positions -1 and +2 refer to bases on the left and right of the boxed base respectively. Olson et al.
Assay Not Sensitive enough High variation between replicate samples Signal inhibition from Total RNA or Cell lysate samples • Incubation time was reduced. • Incubate both the primary and secondary reactions for the recommended times. The secondary reaction time can be increased provided that the background is low. • Design does not include stacker oligo. • Redesign with stacker oligo.
• The primary reaction has an optimum temperature • Verify primary reaction temperature by testing Invader other than 60°C. reactions, including negative controls and 1–2 moles of target, at 60 +/-5°C. If the reaction peak is below 58°C, increase the length of the probe by one base. If the reaction peak is above 62°C, decrease the length of the probe by one base. Alternatively, the primary reaction can be performed at the optimal primary reaction temperature with a 60°C secondary reaction incubation.
• Incomplete mixing. • Thoroughly mix all reagents before dispensing into reaction plate. The secondary reaction mix should be added beneath the overlay.
• Pipeting error. • When using a multi-channel pipet, visually inspect tips when aspirating solution to ensure that reagent volumes are equal in all channels.
• Reaction evaporation. • Overlay the reactions with Chill-out liquid wax or RNase-free mineral oil.
• Total RNA is contaminated with genomic DNA. • Use a RNA isolation method that minimizes the presence of genomic DNA. • Too much total RNA was added to the assay. • Add 0.1 to 200 ng of total RNA per reaction depending on expression level of the gene. Do not add more than 500 ng of total RNA. • Cell lysate preparation contained residual PBS • Remove PBS by gently blotting the tissue culture (>5 µL). plate on absorbent paper or thorough aspiration. • Cells were washed with PBS that contained CaCl2 • Wash cells with PBS that does not contain CaCl2 and MgCl2. and MgCl2. Invader RNA Assay Olson et al. the sample net signal values that fall within the linear range of the assay. For relative quantitation, the no target control is required to determine background signal. Additionally, sample signals can be normalized to an invariant housekeeping gene signal using the biplex format for the Invader RNA assay.
4. Notes
1. Use RNase-free disposables and reagents for sample and reaction preparation.
2. The dynamic range of the assay is typically limited to 2–3 logs when using an endpoint read method. Varying the secondary reaction time, sample concentration or using a real-time fluorescence plate reader can extend the dynamic range of the assay.
3. Agarose gel electrophoresis and ethidium bromide staining can assess the purity and integrity of the RNA sample. Genomic DNA can inhibit signal generation if present at high levels and lead to inaccurate RNA quantitation if using A260 measurements.
4. High background signal can be caused by unpurified probes and FRET oligos. Anion exchange high-performance liquid chromatography is the recommend purification for these oligos. Although we routinely synthesize Invader Assay oligonucleotides at Third Wave Technologies, oligos have been synthesized by commercial suppliers including Qiagen Operon (CA) and BioSearch Technologies Inc. (CA).
5. When preparing cell lysates, be sure to remove residual PBS before lysing the cells because PBS can inhibit the assay. If the lysate samples are generating unusually high signal across the entire plate, the cellular nucleases may not be heat inactivated. Make sure lysates are heated at 75°C for at least 15 min.
6. In vitro transcript dilutions should be stored in tRNA carrier 20 ng/µL (Sigma, cat. no. R-5636) at -20°C or -70°C (for long-term storage).
References
1. de Arruda, M., Lyamichev, V.
2. Hall, J. G., Eis, P. S., Law, S. M., et al. (2000) Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc. Natl. Acad. Sci. USA 97, 8272–8277.
3. Eis, P. S., Olson, M. C., Takova, T., et al. (2001) Nat. Biotechnol. 19, 673–676.
4. Lyamichev, V., Brow, M. A., and Dahlberg, J. E. (1993) Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260, 778–783.
5. Ma, W., Kaiser, M. W., Lyamichev, N., et al. (2000) RNA template-dependent 5′ nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases. J. Biol. Chem. 275, 24693–24700.
6.
Invader RNA Assay
7. Lyamichev, V. I. and Neri, B. P. (2003) Invader assay for SNP genotyping. Single nucleotide polymorphisms (Kwok, P., ed.). Humana,
8. Burczynski, M. E., McMillian, M., Parker, J. B., et al. (2001) Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse transcription polymerase chain reaction and the RNA invasive cleavage assay. Drug Metab.
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10. Allawi, H. T. and SantaLucia, J. Jr. (1997) Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry 36, 10581–10594.
11. Gray, D. M. (1997) Derivation of nearest-neighbor properties from data on nucleic acid oligomers. I. Simple sets of independent sequences and the influence of absent nearest neighbors. Biopolymers 42, 783–793.
12. Mathews, D. H., Burkard, M. E., Freier, S. M., Wyatt, J. R., and Turner, D. H. (1999) Predicting oligonucleotide affinity to nucleic acid targets. RNA 5, 1458–1469.
13. Allawi, H. T., Dong, F., Ip, H. S., Neri, B. P., and Lymichev, V.
14. Lane, M. J. (1997) The thermodynamic advantage of DNA oligonucleotide stacking hybridization reactions: energetics of a DNA nick. Nucleic Acids Res. 25, 611–617.
15. Invader RNA Assay product information sheet for the human IL8 oligonucleotides and control kit, http://www.twt.com/files/15795.lbl.pdf.
Monitoring Eukaryotic Gene Expression Using Oligonucleotide Microarrays Jennifer Lescallett, Marina E. Chicurel, Robert Lipshutz, and Dennise D. Dalma-Weiszhausz
Summary
An increasing number of biological and medical research questions depend on obtaining global views of gene expression. In this chapter, we will describe how oligonucleotide microarrays have been used to accomplish this goal. In particular, we will focus on the use of GeneChip arrays®, which provide high levels of reproducibility, sensitivity, and specificity. Target preparation, hybridization, washing, signal detection, and data analysis will be described in detail. Additionally, we will discuss options for facilitating data sharing, including the creation of databases, and the use of internet tools that help users place their results in the context of data from public and proprietary databases. There is so much interest and innovation in the field of genomics that protocols are constantly evolving. This chapter should be used as a genomic profiling guide only. We urge readers to consult www.affymetrix.com for the most current products and protocols. Key Words: High-density oligonucleotide microarray, DNA microarray, gene expression, expression profiling, genomics