Contributions
Abstract: PB1354
Type: Publication Only
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
Reliance on RNA usage is integral in many scientific fields. For many applications, RNA integrity is required and must be maintained during storage. Current gold standard for RNA storage at -80°C1 may still jeopardize RNA integrity due to ribozyme activity2. An alternative method for RNA preservation is essential in these growing fields, where the use of RNA is imperative. BRS (Biomatrica, San Diego, CA), is a unique RNA preservative that has demonstrated RT stability for up to 12 years in accelerated aging studies3.
Aims
BRS has shown excellent stability at 50°C for total RNA; however, BRS IVT-RNA stability data has not been published. We evaluated preservation of e1a2 IVT-RNA in BRS and CPHD1 for calibration of the Cepheid Xpert BCR-ABL Ultra p190 Prototype Assay, which is designed as a ≤ 2.5 hour test, to monitor minimal residual disease in Ph+ ALL patients.
Methods
e1a2 IVT-RNA was prepared in DEPC-treated water or in CPDH1 at 200pg/µL in E.coli tRNA. 10 µL of the IVT-RNA mixture was added to BRS or CPDH1 and dried in a laminar flow hood for ~24 hours. Completely dried IVT-RNA tubes were placed into moisture barrier bags with desiccant packets, heat sealed, and stored at RT (19°C-23°C) or 50°C. The IVT-RNA in DEPC-treated water controls were aliquoted into microfuge tubes and stored at -80°C. At designated time points, aliquots from each condition were diluted in assay buffer and tested with the Xpert BCR-ABL Ultra p190 Prototype Assay and with digital droplet PCR (ddPCR) on the BioRad QX-200 ddPCR systems (BioRad, Hercules, CA).
Results
Storage of IVT-RNA in BRS at RT and 50°C show excellent stability, comparable to the gold standard (-80°C) through the duration of the study. A general linear model (GLM) was fit to evaluate the effect of time, storage temperature, and IVT-RNA input level on measured e1a2 transcript (a2 Ct). Significant value was set at 0.01, according to Bonferroni correction method. All non-significant two-factor interaction and second-order terms were dropped from the analysis, yielding a general regression equation which predicts an increase of 0.43 Ct over 36 weeks. The reduced model ANOVA table showed no significant difference in e1a2 Ct between storage temperature with p-value = 0.73. E1a2 copy number was monitored in parallel, for the two lowest input levels (0.1fg and 1fg) with ddPCR. A GLM was similarly run to generate a regression equation to predict copy number (CN). Reduced model ANOVA from Design Of Experiment for CN show non-statistically significant for storage temperature with p-value = 0.296. At 36 weeks, e1a2 IVT-RNA performs remarkably well at 50°C, comparable to T0 baseline. Cycle threshold, endpoint fluorescent and CN comparability are indicators of excellent stability (Table 1). Preliminary data for the CPHD1 RNA show promising results after dried storage at RT for 20 days + 16 days at 50°C.
Conclusion
BRS is an effective IVT-RNA storage formula at 50°C for up to the evaluated duration of 36 weeks, equivalent to 5 years at RT based on accelerated aging. Future timepoints up to 24 months will be monitored. The study goal was to determine storage stability of e1a2 IVT-RNA for usage in the Xpert BCR-ABL Ultra p190 Prototype Assay. However, during the initial evaluation, BRS production was discontinued and Cepheid has consequently developed an alternative formula (CPHD1) to store IVT RNA at RT. Preliminary data for CPHD1 show promising results. Further studies will need to be performed to elucidate the effectiveness of the CPHD1 RNA stable reagent for storage at RT.
Keyword(s): Acute lymphoblastic leukemia, BCR-ABL, PCR, Ph+ ALL
Abstract: PB1354
Type: Publication Only
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
Reliance on RNA usage is integral in many scientific fields. For many applications, RNA integrity is required and must be maintained during storage. Current gold standard for RNA storage at -80°C1 may still jeopardize RNA integrity due to ribozyme activity2. An alternative method for RNA preservation is essential in these growing fields, where the use of RNA is imperative. BRS (Biomatrica, San Diego, CA), is a unique RNA preservative that has demonstrated RT stability for up to 12 years in accelerated aging studies3.
Aims
BRS has shown excellent stability at 50°C for total RNA; however, BRS IVT-RNA stability data has not been published. We evaluated preservation of e1a2 IVT-RNA in BRS and CPHD1 for calibration of the Cepheid Xpert BCR-ABL Ultra p190 Prototype Assay, which is designed as a ≤ 2.5 hour test, to monitor minimal residual disease in Ph+ ALL patients.
Methods
e1a2 IVT-RNA was prepared in DEPC-treated water or in CPDH1 at 200pg/µL in E.coli tRNA. 10 µL of the IVT-RNA mixture was added to BRS or CPDH1 and dried in a laminar flow hood for ~24 hours. Completely dried IVT-RNA tubes were placed into moisture barrier bags with desiccant packets, heat sealed, and stored at RT (19°C-23°C) or 50°C. The IVT-RNA in DEPC-treated water controls were aliquoted into microfuge tubes and stored at -80°C. At designated time points, aliquots from each condition were diluted in assay buffer and tested with the Xpert BCR-ABL Ultra p190 Prototype Assay and with digital droplet PCR (ddPCR) on the BioRad QX-200 ddPCR systems (BioRad, Hercules, CA).
Results
Storage of IVT-RNA in BRS at RT and 50°C show excellent stability, comparable to the gold standard (-80°C) through the duration of the study. A general linear model (GLM) was fit to evaluate the effect of time, storage temperature, and IVT-RNA input level on measured e1a2 transcript (a2 Ct). Significant value was set at 0.01, according to Bonferroni correction method. All non-significant two-factor interaction and second-order terms were dropped from the analysis, yielding a general regression equation which predicts an increase of 0.43 Ct over 36 weeks. The reduced model ANOVA table showed no significant difference in e1a2 Ct between storage temperature with p-value = 0.73. E1a2 copy number was monitored in parallel, for the two lowest input levels (0.1fg and 1fg) with ddPCR. A GLM was similarly run to generate a regression equation to predict copy number (CN). Reduced model ANOVA from Design Of Experiment for CN show non-statistically significant for storage temperature with p-value = 0.296. At 36 weeks, e1a2 IVT-RNA performs remarkably well at 50°C, comparable to T0 baseline. Cycle threshold, endpoint fluorescent and CN comparability are indicators of excellent stability (Table 1). Preliminary data for the CPHD1 RNA show promising results after dried storage at RT for 20 days + 16 days at 50°C.
Conclusion
BRS is an effective IVT-RNA storage formula at 50°C for up to the evaluated duration of 36 weeks, equivalent to 5 years at RT based on accelerated aging. Future timepoints up to 24 months will be monitored. The study goal was to determine storage stability of e1a2 IVT-RNA for usage in the Xpert BCR-ABL Ultra p190 Prototype Assay. However, during the initial evaluation, BRS production was discontinued and Cepheid has consequently developed an alternative formula (CPHD1) to store IVT RNA at RT. Preliminary data for CPHD1 show promising results. Further studies will need to be performed to elucidate the effectiveness of the CPHD1 RNA stable reagent for storage at RT.
Keyword(s): Acute lymphoblastic leukemia, BCR-ABL, PCR, Ph+ ALL