Lumit® dsRNA Detection Assay
Assay to Detect and Quantify Double-Stranded RNA Contamination in IVT mRNA Samples
- Specific: No cross reactivity with ssRNA or DNA
- Quantitative: Not dependent on sequence or size (≥30bp)
- Simple Workflow: No time-consuming wash steps or immobilization
- Novel Approach: Does not utilize antibodies to detect dsRNA
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$ 1,320.00
Catalog Number: W2041
$ 5,610.00
Catalog Number: W2042
Catalog Number: W2010
Catalog Number: W2040
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Quantify dsRNA with Confidence
High-quality in vitro transcription (IVT) products are crucial for research and RNA therapeutic development. However, IVT can generate double-stranded RNA (dsRNA) byproducts. This contaminant can have significant negative downstream effects on drug substances and products, such as high immunogenicity. Sensitive and quantitative assessment of dsRNA is critical to control and optimize mRNA therapeutic and vaccine manufacturing processes.
The Lumit® dsRNA Detection Assay is a novel technique to quantify dsRNA in a sample. Lumit® Assay technology is based on NanoBiT® luciferase complementation. NanoBiT® subunits, SmBiT and LgBiT, are genetically fused to dsRNA binding proteins. In the presence of dsRNA, the SmBiT and LgBiT subunits are brought together to form an active NanoBiT® Luciferase, generating a luminescent signal proportional to the amount of dsRNA in a sample. The result is a concentration-dependent luminescent signal for quantitative and specific measurement of dsRNA.
Simple Protocol Streamlines dsRNA Quantification
The assay is solution-based and homogeneous:
- No wash or sample transfer steps needed
- Amenable to high-throughput applications
Size and Sequence-Independent dsRNA Assay: The Lumit® dsRNA Detection Assay can sensitively quantify dsRNA independent of sequence and size.
Better Performance than Commercial ELISA Kits: The Lumit® dsRNA Detection Assay provides an accurate linear range of 0.04 to 2.5ng/ml. Commercial dsRNA ELISA kits do not accurately quantitate dsRNA at these concentrations.
Detect and Quantify dsRNA in Complex IVT Samples: The Lumit® dsRNA Detection Assay can accurately quantify dsRNA in IVT samples with mixed RNA species.
Detecting dsRNA
The Lumit® dsRNA Detection Assay exhibits outstanding intra- and inter-assay precision and extremely low limit of detection.
Standard response curve generated using the Lumit® dsRNA Detection Assay across a series of dsRNA concentrations. Each point represents the mean of six replicates, and the data were collected from three independent experiments. Measurements were recorded using a GloMax® Discover plate luminometer. The assay demonstrated a calculated Limit of Detection (LoD) of 0.37pg/ml and a Limit of Quantitation (LoQ) of 1.12pg/ml, highlighting its exceptional sensitivity and reproducibility.
|
Intra-Assay Precision |
|||
| Expected (ng/ml) |
Apparent n=16 (ng/ml) |
Recovery n=16 (%) |
CV (%) |
| 0.125 | 0.133 | 106 | 3.2 |
| 0.5 | 0.507 | 101 | 4.4 |
| 2.0 | 2.070 | 104 | 3.6 |
| Average: 3.7 | |||
Three concentrations of dsRNA were tested, each as replicates of 16. Average apparent concentrations were determined by linear interpolation using the standard curve.
|
Inter-Assay Precision |
|||
| Expected (ng/ml) |
Apparent n=48 (ng/ml) |
Recovery n=48 (%) |
CV (%) |
| 0.125 | 0.130 | 104 | 6.2 |
| 0.5 | 0.494 | 99 | 5.2 |
| 2.0 | 2.069 | 103 | 3.6 |
| Average: 5 | |||
Three concentrations of dsRNA were tested as replicates of 16 in three experiments. Average apparent concentrations were determined by linear interpolation using the standard curve.
Detecting Modified and Unmodified dsRNA with Lumit® dsRNA Assay
Performance of the Lumit® dsRNA Detection Assay with dsRNA standards containing various nucleotide modifications. Each dsRNA standard (unmodified, pseudouridine, N1-methylpseudouridine, 5-methoxyuridine and 5-methylcytidine) was HPLC-purified to equivalent purity and tested in three independent experiments. Responses were normalized to the unmodified dsRNA standard using linear fits of the resulting curves. The assay demonstrated linear detection ranges across all modified and unmodified standards, with minimal variability. Note: For the most accurate quantitation of samples containing modified nucleotides, it is recommended to use a standard containing the same modified nucleotide as that present in the sample.
|
RNA Modification |
Average |
| Unmodified (contained in assay kit) | 100 |
| Pseudouridine (Ψ) | 77.2 |
| N1-methylpseudouridine (m1Ψ) | 65.8 |
| 5-methoxyuridine (5moU) | 30.6 |
| 5-methylcytidine (5meC) | 102.5 |
Specific Detection of dsRNA in IVT Products
The Lumit® dsRNA Detection Assay can accurately quantify dsRNA in IVT samples with mixed RNA species.
Lumit® dsRNA Detection Assay quantification of dsRNA in IVT samples with mixed RNA species. IVT samples were treated ± RNase If (ssRNA-specific) or RNase III (dsRNA-specific); total RNA (QuantiFluor® RNA) and dsRNA (Lumit®) were quantified.
- Left: Digestion of ssRNA with RNase If significantly reduced total RNA but did not impact the amount of dsRNA detected.
- Right: Digestion of dsRNA with RNase III into fragments ≤20bp results in a complete loss of activity in the Lumit® dsRNA Detection Assay.
Protocols
Complete Protocol
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
Lumit® Detection Substrate B |
VB405A | 1 × 160μl |
dsRNA Assay Buffer (5X) |
W201A | 1 × 8ml |
Lumit® dsRNA Sensor-LgBiT |
W202A | 1 × 15μl |
Lumit® dsRNA Sensor-SmBiT |
W203A | 1 × 15μl |
dsRNA Standard |
W204A | 1 × 15μl |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
What's in the box?
| Item | Part # | Size |
|---|---|---|
Lumit® Detection Substrate B |
VB405A | 5 × 160μl |
dsRNA Assay Buffer (5X) |
W201A | 5 × 8ml |
Lumit® dsRNA Sensor-LgBiT |
W202A | 5 × 15μl |
Lumit® dsRNA Sensor-SmBiT |
W203A | 5 × 15μl |
dsRNA Standard |
W204A | 5 × 15μl |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.What's in the box?
| Item | Part # | Size |
|---|---|---|
dsRNA Assay Buffer (5X) |
W201A | 1 × 8ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
What's in the box?
| Item | Part # | Size |
|---|---|---|
dsRNA Standard |
W204A | 1 × 15μl |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
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