This document introduces a method for isolating total RNA samples using the Agilent Total RNA Isolation Kit and the Agilent DNase Accessory Kit, which can effectively reduce the genomic DNA contamination in total RNA samples.
This document presents an application note comparing the Agilent Total RNA Isolation Mini Kit with a leading silica column-based kit for isolating total RNA from mouse tissues. The study highlights that while RNA integrity is crucial, the presence of genomic DNA (gDNA) can lead to false positive signals in gene expression analysis methods like real-time PCR and microarray hybridization.
The authors utilized quantitative real-time PCR to measure gDNA contamination levels in RNA samples isolated from mouse thymus, spleen, and pancreas. Results showed that the Agilent kit alone provided up to 1000-fold lower gDNA contamination compared to the silica-based method. When combined with the new Agilent DNase Accessory Kit, the method achieved the lowest levels of contamination, reaching as low as 0.0003% for pancreas tissue.
| Product Number (RNA Kit) | 5185-6000 |
|---|---|
| Product Number (DNase Kit) | 5188-2735 |
| Tissue Weight (Thymus/Spleen) | 15 mg |
| Tissue Weight (Pancreas) | 10 mg |
| Target Gene | mouse GAPDH gene |
| Fragment Size | 79 bp |
The Agilent method results in up to 1000-fold lower levels of gDNA contamination compared to a silica column-based method.
The lowest level of gDNA contamination achieved was 0.0003% for pancreas tissue when using the Agilent kit with the DNase Accessory Kit.
Primers and probes specific to the mouse GAPDH gene were used to detect gDNA contamination.