Over the last two decades, an international study designed to study glaucoma in Africa necessitated the collection of thousands of blood samples from study participants in the West African country of Ghana, for the purpose of DNA extraction
. During one shipment of several hundred samples to the Duke University Biobank in Durham, North Carolina, the samples experienced high temperatures and were found to be hemolyzed when they arrived at our facility. The degree of hemolysis raised concerns about the integrity of the samples, the effect on potential DNA yield, as well as their usability for further analyses.
At our biobanking and DNA extraction facility we sought to isolate genomic DNA at an optimal yield. We initiated the study using the Autopure LS® (AutoGen) purification protocol on a subset of the samples; however, the genomic DNA yield was too low for subsequent analyses in the majority of extracted samples. We decided to investigate a different commercial system based on an alternative purification protocol. The ReliaPrep Large Volume HT gDNA Isolation System (Promega) was used as a comparison on the same subset of samples processed using the Autopure protocol. We demonstrate here that the ReliaPrep purified samples consistently generated significantly higher quantities of genomic DNA. A comparison of DNA size distribution by agarose gel electrophoresis also demonstrated a more favorable outcome using the ReliaPrep system.
Materials & Methods
Compromised Whole Blood Protocol on the Autopure LS® (AutoGen): Whole blood tubes (PAXGene) were thawed and transferred to Autopure conical tubes. The Autopure dispensed 30–35ml red blood cell (RBC) Lysis Solution (Reagent 1) into each input tube. The system uses Reagent 1 to balance the tubes before centrifugation. The amount dispensed into each tube varies depending on the initial sample volume. The total volume of sample and Reagent 1 was 40ml. Samples were incubated in RBC Lysis Solution for 5 minutes 30 seconds to lyse the red blood cells. The samples were rotated gently to mix during incubation. Samples were then centrifuged at 3000 x g for 2 minutes to pellet the white blood cells, and the supernatant was discarded. Then, 4ml Autopure Precipitation Solution (Reagent 3) and 10ml Autopure Cell Lysis Solution (Reagent 2) were dispensed into each input tube followed by mixing the samples vigorously for 2 minutes to lyse the cells and precipitate the proteins. Samples were centrifuged at 3000 x g for 5 minutes to precipitate proteins into a tight pellet at the bottom of the input tube. The DNA-containing supernatant from step 4 was transferred into the output tubes that contained 12ml Autopure 100% Isopropanol. Samples were rotated gently 50 times to precipitate the DNA, then centrifuged at 3000 x g for 5 minutes to pellet the DNA. The isopropanol supernatant was removed, and the output tubes were inverted for 1 minute to evaporate any remaining alcohol. The final wash was 12ml Autopure 70% Ethanol (Reagent 5) in the output tubes followed by centrifugation at 3000 x g for 5 minutes to pellet the DNA. The ethanol supernatant was removed and the tubes inverted for 1 minute to evaporate any remaining alcohol. Finally, 1ml of Autopure DNA Hydration Solution (Reagent 6) was dispensed into the output tubes to rehydrate the DNA. After the Autopure had completed its run, output tubes were incubated at 65°C for 1–2 hours to dissolve the DNA, followed by incubation at room temperature (15–25°C) overnight, with gentle shaking.
ReliaPrep Large Volume HT gDNA Isolation System (catalog #2751) using a HSM 2.0 Instrument (catalog #2715): Whole blood tubes were thawed and transferred to 50ml conical tubes with an average sample volume of approximately 6ml. A 0.14ml volume of Proteinase K was added to each sample, and they were mixed at 500 rpm for 1 minute. Then, 0.87mL of Alkaline Protease was added and samples were mixed at 500 rpm for 1 minute. Samples had 7ml of Lysis Buffer added and were then incubated at 65°C for 30 minutes with shaking at 500 rpm, followed by 10 minutes of shaking at 500 rpm without heat. An 8.4ml volume of Binding Buffer was added to each sample, followed by shaking samples at 550 rpm for 3 minutes. After shaking, 0.7ml of thoroughly resuspended ReliaPrep Resin was added to the samples. Samples were shaken at 550 rpm for 20 minutes, then the HSM 2.0 Instrument collected the ReliaPrep Resin at the side of the tube for 20 minutes. When prompted by the instrument, the lysate was slowly aspirated and discarded. After the initial removal, an additional aspiration was performed to ensure that all lysate was removed, then 7.3ml of Prepared Wash Buffer was added to each tube, and they were then shaken at 500 rpm for 2 minutes. Next, samples were mixed via pipetting at least 10 times to thoroughly disperse the resin. The samples were again shaken for 2 minutes to wash the resin. The HSM 2.0 Instrument collected the ReliaPrep Resin for 3 minutes, then the fluid from each tube was slowly vacuum aspirated and discarded. After the initial removal, an additional aspiration was performed to ensure that all Wash Buffer was removed. An additional 7.3ml of Prepared Wash Buffer was added to each tube and samples were shaken at 500 rpm for 3 minutes, followed by 3 minutes of shaking at 700 rpm, then the HSM 2.0 Instrument collected the ReliaPrep Resin at the side of the tube for 3 minutes. Samples were slowly aspirated from each tube. After removal, an additional aspiration was performed to ensure that all Wash Buffer was removed and discarded. Next, 6.3 ml of 50% ethanol was added to each sample, and they were shaken at 500 rpm for 4 minutes. The HSM 2.0 Instrument collected the ReliaPrep Resin at the side of the tube for 3 minutes, then the ethanol was slowly aspirated from each tube. After the initial removal, an additional aspiration was performed to ensure that all ethanol was removed. Then, 1ml of nuclease-free water was added to each tube followed by shaking for 3 minutes at 600 rpm. Samples were then incubated at 70°C for 20 minutes with shaking at 400 rpm. The HSM 2.0 Instrument collected the ReliaPrep Resin at the side of each tube for 5 minutes, then the eluate was transferred to a 1.5ml tube.
The DNA concentration of each sample was assessed using PicoGreen methodology, a fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in solution. DNA yields for nearly every sample were higher for the ReliaPrep system, compared with the Autopure protocol. Figure 1 shows the difference in genomic DNA yield for each sample; the value shown represents the difference in yield between the two methods (yield obtained using the Autopure method subtracted from the yield obtained for that same sample using the ReliaPrep method). The average genomic DNA yield for 406 samples extracted with the ReliaPrep method was approximately 40 µg per sample, compared to an average yield of approximately 9 µg per sample using the Autopure method.
Figure 1. ReliaPrep Minus Autopure Genomic Yields
The comparative yield for each sample using the two extraction methods was calculated by subtracting the amount recovered by the Autopure purification from the amount recovered using the ReliaPrep purification system. Most samples show a significantly higher yield when purified using ReliaPrep.
Once it became clear that the ReliaPrep system was superior, an additional 213 samples were extracted with this method, with an average yield of 62.7 µg of DNA (no comparison to the Autopure method for these 213 samples). Overall, it is critical to note that the majority of the samples analyzed in this study were selected for this comparison because they provided poor (or no) DNA yields using the Autopure protocol. We were not surprised by the preliminary results showing low or no genomic DNA yield, as these samples had been overheated during shipment. However, the ReliaPrep method allowed for efficient purification of genomic DNA. We recommend that researchers working with compromised blood samples consider the increased yield obtained through extraction with the ReliaPrep system.
In summary, the Autopure and ReliaPrep systems were used to purify genomic DNA, and a direct comparison demonstrates that the ReliaPrep protocol resulted in significantly higher yields of DNA. The yield as well as the quality of the genomic DNA isolated from these irreplaceable samples using Reliaprep allows for additional and potentially valuable studies. The results presented here suggest that for blood samples designed “precious” or potentially compromised because of their history and the method in which they were collected, stored, handled and/or shipped to another location for future applications, the Reliaprep Large Volume HT gDNA Isolation System is an excellent choice for genomic DNA purification. This system holds promise for researchers wishing to work with blood samples that cannot be collected again but may hold critical information pertaining to their research initiatives.