1Omega Bio-tek, Inc, Norcross GA 30071
Materials and Methods
Results and Discussion
The purified DNA yield from the Salivagard® HT DNA samples are shown in Figure 1. The differences in the DNA yield can be attributed to inter-donor variability. The average A260/A280 ratio was ~1.7±0.1 indicating good quality of extracted DNA. TapeStation® analysis was performed on the purified DNA to determine the size of the genomic DNA that was extracted as well as its integrity. TapeStation® 2200 analysis software automatically calculated each sample’s DNA Integrity Number (DIN). It typically ranges from 0 to 10 (10 being highest integrity) and is determined on the basis of genomic DNA sample fragmentation. Figure 2 shows the TapeStation® analysis performed on the extracted DNA from the saliva samples stored in Salivagard® HT DNA tubes. The purified DNA across the 8 samples migrated as a well-defined band without fragmentation and was above the largest ladder peak (48,500 bp), with TapeStation® software analysis determining it to be over 60 kb. The DIN values ranged between 7.8-9.3, suggesting high-quality, intact DNA with minimal degradation.The quality of the DNA obtained from each extraction was determined based on the Ct values generated from a real-time PCR reaction. Figure 3 shows the average Ct values obtained on 10-fold and 100-fold dilutions of the purified DNA using human-specific primers. The Ct values across all the dilutions indicate positive amplification and correspond well with the yields shown in Figure 1. Typically, Ct values of samples whose concentration differs by a factor of 10 are 3.3 cycles apart. The average ΔCt value between the 100-fold and 10-fold was ~3.5 indicating no inhibition.
Our results demonstrate that DNA obtained from saliva samples collected and stored in Biomatrica’s Salivagard® HT DNA tubes is of high-quality and integrity. Omega Bio-tek has developed a rapid, reliable, fully automated solution by translating their chemistry onto the Hamilton’s Microlab® STAR™ platform that can extract 96 samples in less than 90 minutes. Biomatrica’s Salivagard® HT DNA tubes together with the Omega/Hamilton protocol provide a complete workflow solution from sample collection and storage through processing and purification that is user-friendly, simple and fast.
Figure 1. DNA was extracted from 250 μL of saliva stabilized in Salivagard® HT DNA tubes using Omega Bio-tek’s Mag-Bind® Blood & Tissue DNA HDQ 96 Kit (M6399). The eluted DNA was quantified using Promega’s QuantiFlour® dsDNA system.
Figure 2. TapeStation® analysis performed on the extracted DNA from 250 μL of saliva stabilized in Salivagard® HT DNA tubes.
Figure 3. Average Ct values obtained amplifying the purified DNA from stabilized saliva using Omega Bio-tek’s kit.