During the process of preparing mRNA using T7 RNA polymerase In Vitro Transcription (IVT), by-products such as double-stranded RNA (dSRNA) are generated. dSRNA not only triggers the body's innate immune response, It may also lead to the degradation of mRNA, so the residual amount of dsRNA is a key quality attribute of mRNA drugs.
This kit employs a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect the content of dsRNA in in vitro transcription systems or synthesized mRNA stock solutions. The microplate is coated with capture antibody to form solid-phase antibody. dsRNA standard and sample to be tested are added to the solid-phase antibody microplate. Then, horseradish peroxidase (HRP) -labeled detection antibody is added to form antibody-antigen-antibody complex. After thorough washing, substrate TMB is added for color development. The chromogenic solution is converted into blue under the catalysis of HRP enzyme and eventually into yellow under the action of acid. The depth of the color is positively correlated with the amount of dsRNA in the sample.
Product Name: T7 High Efficiency Transcription Kit Catalog Number: BP-01-50
Product Name: T7 RNA Polymerase 3.0 Item Number: BP-E11
Product Name: PolyA Tail Length Distribution Detection Pretreatment Kit Item Number: BP-11-10
1. Standard curve
Description: A standard curve was established by gradient dilution of double-stranded RNA standards.
Figure 1 shows the absorption values of dsRNA standards (left) and the prepared standard curves
Conclusion：Double-stranded RNA standards are used for absorbance determination after gradient dilution. The data was processed using a four-parameter fitting curve. The results show that the value of R² is greater than 0.99.
2. Recovery rate of the reagent kit
Description：Different concentrations of dsRNA standards were added to samples of known concentrations. The accuracy of the kit recovery rate was determined based on the difference between the measured concentration and the theoretical concentration.
Table 1 Recovery Rate of Reagent Kits
Conclusion：The relative deviations of the sample detection concentrations were all ≤15%, and the recovery rates conformed to 80%-120%.
3. Repeatability
Description：Six-well determination was conducted on high, medium and low concentration samples respectively, and the detected concentration CV met the requirements.
Table 2 Repeatability Verification
Conclusion：Repeatability tests were conducted at three concentrations (high, medium and low) of standard samples. Each group of experiments was repeated six times. The coefficient of variation of each group was less than 10%, meeting the requirements and indicating that the repeatability of this kit was good.
4. Inter-batch precision
Description：The same operator tested three batches of reagent kits at the same time. For each batch, high, medium and low concentration samples were tested six times each. A total of 54 test results were obtained for samples of all concentrations, and the precision results met the requirements.
Table 3 Inter-batch Precision
Conclusion：The accuracy was evaluated by the same operator through a single analysis of three batches of test kits. Each batch of reagent kits is tested six times with high, medium and low concentration samples respectively. The coefficient of variation of all batches was less than 10%, within the preset acceptable range, indicating good inter-batch precision.
5. Human precision
Description：Two operators simultaneously tested one batch of reagent kits. For each batch, they tested high, medium and low concentration samples six times each, with a total of 36 test results for samples of all concentrations. The precision results met the requirements.
Table 4 Human Precision
Conclusion：The assessment method for artificial precision is as follows: Two operators simultaneously test the same batch of reagents. Each operator conducts six repeated determinations on the high-concentration, medium-concentration and low-concentration samples respectively. The obtained precision values are all within the preset acceptable range.
