dsRNA (IVT) assay details

dsRNA (IVT) kit standard curve

Intra-assay precision table

Each of the 3 samples was measured 24 times, and the % CV was calculated for each sample.

Inter-assay precision table

Each of the samples was measured in 3 independent experiments performed by different operators. The % CV was calculated for each sample.

Dilutional linearity table

Samples were generated from serial dilution of dsRNA in diluent#14. The excellent % of recovery obtained from these experiments shows the good dilution linearity of the assay (Dilution test acceptance criteria: 85-115%).

dsRNA (IVT) Kit cross-reactivity table

Cross reactivities were assessed using other reagents commonly present in mRNA samples during IVT production process, such as single strand RNA (ssRNA), DNA template (circular and fragments) or T7 polymerase. Titration of each reagent diluted in the kit diluent was performed using concentrations in line with what if found in IVT production processes.

For each reagent and concentration tested, 10 µL of each sample was transferred into a white detection plate (384 low volume), and 5 µL of each HTRF dsRNA (IVT) detection reagents were added. The HTRF signal (Delta Ratio) was recorded after overnight incubation at room temperature (22°C).

No detection signal was recorded using for any of the ssRNA, DNA Template and T7 polymerase samples in comparison with positive controls of dsRNA. In these assay conditions, the kit shows detection specificity for dsRNA over other tested IVT reagents.

dsRNA (IVT) Kit interference using other commonly used IVT reagents

To test for the absence of interference of this dsRNA (IVT) kit with other reagents commonly used in mRNA IVT process, a fixed concentration of a 142 bp natural dsRNA sample (50 ng/mL) was mixed with a 2000-folds more concentrated 142 bp ssRNA sample (10 µg/mL), digested DNA template (10 µg/mL) and 500 U/mL of T7 polymerase. A dsRNA sample at 50 ng/mL was used as reference control.

10 µL of each sample were plated in a 384sv white plate and 10 µL of HTRF (IVT) kit detection reagents mix were added. The HTRF signal (Delta Ratio) was recorded after overnight incubation at room temperature (22°C).

No significant change of HTRF signal was recorded between the natural dsRNA control and the dsRNA + IVT reagent mix, demonstrating the absence of interference from common reagents present in IVT samples.

Role of standard type in dsRNA sample measurement

The HTRF dsRNA (IVT) kit includes a natural dsRNA standard for more relevant quantification of dsRNA in real IVT samples compared to synthetic Poly-IC standards, which are commonly used in Dot blot or ELISA dsRNA assays. A synthetic Poly IC standard, part of HTRF viral dsRNA kit (64RNAPEG), is also available as spare reagent (#64RNATDA) and is compatible with this HTRF dsRNA (IVT) kit.

We present here standard curves using the natural dsRNA standard from the HTRF dsRNA (IVT) Kit and the synthetic Poly-IC standard (#64RNATDA) from the HTRF dsRNA (virology) Kit. The main result is a 4X higher sensitivity for Poly-IC synthetic standard compared to the dsRNA natural standard. This result is expected and reported in the literature as a consequence of different antibody affinities for natural and synthetic dsRNAs, which is especially true and published for anti-dsRNA gold standard antibodies. This difference of sensitivity between standards is key as it drastically affects the reading of samples when related to the resulting standard curves. The most accurate measurement of dsRNA in a sample is obtained when the standard used is in line with the sample, meaning that synthetic dsRNA standard generally introduce a bias when working with IVT samples that are based on natural nucleic acid.

To demonstrate dsRNA sample quantification correlation using both standards, a natural 100 bp dsRNA (Nterm of GFP protein sequence) was serially diluted in the kit diluent. 10 µL of each sample were added in a 384sv white plate, and 10 µL of HTRF dsRNA (IVT) detection reagents mix was added. The HTRF signal (Delta Ratio) was recorded after an overnight incubation at room temperature (22°C). dsRNA quantification was obtained by interpolating samples signals on each standard curves. Obtained concentrations using each natural and synthetic standard are plotted and presented in the correlation graph.

Results show perfect correlation of natural dsRNA sample quantification using natural dsRNA kit standard and synthetic poly-IC standard.

While the correlation between natural and synthetic dsRNA standard is assured, we still report a difference of 3,5-fold in dsRNA sample quantification between both standards. Due to that bias coming from the synthetic Poly-IC standard, dsRNA sample concentration is underestimated in that case.

different length of dsRNA detectable table

In IVT processes, dsRNA contaminants present themselves as an arrays of fragments of different length in bp. To assess the HTRF dsRNA (IVT) kit’s ability to detect these dsRNA fragment of various length, serial dilutions of different lengths of dsRNA were made in diluent 14. All fragments were generated from the N-term portion of GFP protein sequence.

10 µL of each sample were plated in a 384sv white plate, and 10 µL of HTRF dsRNA (IVT) detection reagents mix were added. The HTRF signal was recorded after overnight incubation at room temperature (22°C).

For this specific sequence, results show that the detectable range of dsRNA fragment length goes from 35 to at least 1000 bp, where green boxes represent range of detection for each dsRNA length. As expected, detection and sensitivity are enhanced when dsRNA length increases, due to the repeat epitope-based detection of the assay.

HTRF dsRNA (IVT) assay compared to Dot Blot

Dot blot assays are commonly used to detect dsRNA particles in samples resulting from In Vitro Transcription (IVT) of mRNA. A comparison between HTRF and Dot Blot was performed to assess the limit of sensitivity of each method.

For HTRF, serial dilutions of dsRNA were made using diluent 14, and 10 µL of each dilution were transferred into a low volume white microplate before the addition of 10 µL of Anti dsRNA detection reagents.

For Dot Blot assays, serial dilutions of the same dsRNA were blotted at 5 µl per drop.

After incubating with primary dsRNA-specific monoclonal J2 antibody and secondary HRP antibody, the membrane was treated with ECL reagent and revealed at 1 sec exposure.

A side-by-side comparison of Dot Blot and HTRF demonstrates that the HTRF assay is 400-fold more sensitive than the Dot Blot, at least under these experimental conditions.

Beyond this difference in sensitivity, it is worth noting than a Dot blot approach to dsRNA detection is generally more qualitative than quantitative.