AQUAPRESERVE

Preserve. Stabilize. Extract. Streamlining Biospecimen Workflow.

AquaPreserve™ is a multifunctional aqueous reagent for DNA/RNA/protein preservation and extraction. It may be used to streamline biospecimen collection, stabilization, transport, storage, distribution, and DNA/RNA/protein extraction. By streamlining the entire biospecimen workflow, AquaPreserve can reduce pre-analytical variability, increase data reproducibility and reliability. AquaPreserve can stabilize and extract total DNA/RNA/proteins from whole blood, plasma, saliva and other liquid or solid biospecimens. AquaPreserve is the only reagent that can extract intact RNA from frozen whole blood samples collected in common anticoagulants.

AquaPreserve Brief Protocol

(Download AquaPreserve User Manual for protocols using AquaPreserve in specimen biobanking, DNA/RNA/protein extraction from fresh or frozen whole blood, buffy coat, plasma, automated DNA/RNA extraction, and more)

1. Lyse the blood cells

Add 0.25 ml of AquaPreserve to 0.25 ml of fresh or frozen whole blood in a 1.5-ml microfuge tube. Vortex to thaw the blood (Do not thaw frozen blood without mixing with AquaPreserve or the RNA will be degraded during blood thawing. However, for blood DNA extraction only, the blood sample should be thawed and incubated at 22 °C for 20 min to degrade the RNA prior to mixing with AquaPreserve.). Incubate at 22 °C for 15 min. Shake the tube vigorously to break up the blood clot and centrifuge at 12,000 xg for 5 min to pellet the debris.

2. Pellet the proteins

Add 0.125 ml of ProSink (#9030, order separately) to the crude lysate. Invert the tube and touch vortex a few times to mix well. Incubate at 22 °C for >30 min (Blood DNA is now stable at 4-22 °C for months, and blood RNA is stable at 4 °C for 2 weeks and at 22 °C for 7 days). Centrifuge at 12,000 xg for 5 min to pellet the proteins.

3. Pellet the DNA/RNA

Transfer the supernatant (~0.7 ml) to a new 1.5-ml microfuge tube. Add 0.9 vol (~0.63 ml) of isopropanol. Touch vortex a few times to mix well. Centrifuge at 12,000 xg for 5 min to pellet the DNA/RNA. Decant to discard the supernatant.

4. Rinse the DNA/RNA pellet

Gently fill up the tube and its lid with 70% ethanol from a squirt bottle and then decant to discard the ethanol solution. Repeat the ethanol rinse once. Tap the tube on a paper towel to remove residual ethanol and let the DNA/RNA pellet air-dry for 5-10 min.

5. Solubilize the DNA/RNA pellet

Add 100 µl of deionized water to the DNA/RNA pellet. Vortex and/or pipet to solubilize the DNA/RNA. Incubate at 22 °C for 10 min. Centrifuge again to pellet any insoluble and transfer the clear DNA/RNA solution to a new tube. Store at –20 °C.

FAQ about AquaPreserve

1. How should I store the AquaPreserve solution?

It may be stored at 22 °C for 12 months. If AquaPreserve becomes precipitated when exposed to low temperature, you may incubate it at 37 °C for 15-20 min to resolubilize the reagent.

2. Can we use AquaPreserve to preserve solid tissue samples?

Yes, you may preserve solid tissue samples in 2 volumes of AquaPreserve for biobanking. The tissue/organ may be immersed in AquaPreserve to keep its gross anatomy, or homogenized and aliquoted for storage and distribution to researchers for DNA/RNA/protein extraction. ProSink is not needed for DNA/RNA extraction from most solid tissues other than the RNase-rich liver and spleen tissues.

3. How should I remove the genomic DNA from the DNA/RNA preparation?

You may add 0.2 U of DNase I to 10-20 μl of DNA/RNA solution in 0.5-1x DNase buffer, and incubate at 22-37 °C for 20-30 min. Then run the digested sample in a 0.8% native agarose gel to confirm the completion of DNA digestion. To inactivate the DNase I, you may use Ambion’s DNase removal reagent or inactivate the DNase I at 65 °C for 15 min.

4. Why did my DNA/RNA solution show a strong absorption below A260?

It is likely due to trace amount of guanidine salt contamination. If it interferes with your downstream applications, you may further purify the extracted DNA/RNA with a silica spin column (e.g., a plasmid miniprep column). Simply add an equal volume of 4 M GuHCl and 1M NaOAc (pH unadjusted, ~7.0) to your DNA/RNA solution and load it into the spin column, centrifuge to allow DNA/RNA binding to the silica membrane, wash the column with 0.5 ml 75% EtOH, and elute the DNA/RNA in 50 μl of deionized water or TE buffer.

5. Can I do RT-PCR without removing the contaminating genomic DNA?

Complete DNA removal may be difficult or unnecessary if you use intron-spanning primers for the PCR amplification. You may also design and use a 5’ tailed RT primer to make the cDNA and then use a pair of PCR primers with one of them complementary to the unique tailed region of the RT primer to amplify the cDNA [Hurteau and Spivack. mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts. Anal Biochem. 2002 Aug 15;307(2):304-15; and Chen, et al. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Research 2005 33(20):e179], especially when intron-spanning is unavailable. In any case, you should always include a no-RT control in your amplification to confirm that your primers do not amplify the contaminating genomic DNA.