The Plasmid Reality
PROTOCOL
Product User Manuals (PDF)
- AquaGenomic User Manual Protocols for genomic DNA extraction
- AquaBluer User Manual Protocols for cell viability, cell proliferation, and cytotoxicity assays
- AquaPlasmid User Manual Protocols for plasmid DNA extraction
- AquaPreserve User Manual Protocols for blood DNA, RNA, and protein preservation and extraction
- AquaRNA User Manual Protocols for concurrent DNA, RNA and protein extraction
Full immersion
Thank you for your interest in our reagent products. Our research reagents are aqueous solution-based and scalable. They are multifunctional, inexpensive and simple to use. Instead of needing 6-8 components, our kits consist of only 1 or 2 components. For example, our AquaPlasmid solution may be used to suspend the bacterial pellet, lyse the bacteria, extract the plasmid DNA and remove the cell debris in a single tube. It is the most economic ($0.5/miniprep) and environment-friendly (nontoxic) plasmid purification product on the market. You may click at the links below to learn more about our products.
AquaBluerTM
Assaying cell viability and cytotoxicity:
Colorimetric or fluorescent
Nontoxic, one-step assay
Costs 26 times less than MTT
AquaGenomicTM
Extracting genomic DNA from all types of specimens:
Scalable
No organic extraction
No column purification
AquaPreserveTM
Extracting DNA and RNA from whole blood:
Fresh or frozen whole blood
Recover total blood DNA
Recover total blood RNA
AquaRNATM
Extracting DNA/RNA/proteins from the same specimens:
Use no phenol/chloroform
Scalable
Maximize specimen value
AquaStoolTM
Extracting fecal and soil DNA and RNA:
Preserve fecal samples
Remove PCR inhibitors
Extract fecal DNA and RNA
AquaPlasmidTM $0.5/miniprep
Isolating and purifying plasmid DNA:
Single-solution
Nontoxic
$0.5/miniprep, lowest cost
Experience a new world
AQUAGENOMIC
AquaGenomicTM is an aqueous reagent for DNA extraction. It may be used to extract DNA from all types of specimens, from bacteria to animal tissues. The extraction protocols are simple, fast, and scalable. AquaGenomic is nontoxic and its lysate may be used for PCR without further DNA purification. Most remarkably, AquaGenomic is highly efficient in extracting DNA from dried specimen swabs. It enables the use of cotton swabs for specimen collection, transportation, and storage at room temperature, therefore, making dried specimen swabs ideal for low-complexity and low-cost biobanking, biosurveillance, and epidemiological research applications.
Features
Comparison
Ordering Information
Brief Protocol
Instruction Manual
FAQ
Use of AquaGenomic for total blood DNA biobanking
ag.png
Features
Innovative
AquaGenomic is a multi-functional aqueous solution for genomic DNA isolation and purification. It achieves cell lysis, DNA extraction, and debris removal in a single step.
Simple
Add AquaGenomic solution to lyse the cells, extract the DNA, and precipitate the cell debris. The crude lysate may be used directly for PCR analysis without further DNA purification.
Easy
No columns, no resins, no slurries, no beads, no filters, no membranes, no needles, no cartridges, no manifolds, no multiple solutions and buffers.
Nontoxic
No phenol, no chloroform, no isoamylacohol, no saturated butanol, no guanidine hydrochloride.
Flexible
One AquaGenomic Kit can be used to isolate DNA from cultured cells, buccal swabs, blood, bacteria, animal or plant tissues. No need to purchase different kits for different starting materials.
Scalable
One single AquaGenomic Kit does all: mini, midi, or maxi. No need to purchase separate mini, midi, or maxi kits.
High yield
About 5-10 ug of DNA can be isolated from 1-2 million cultured cells, 15 mg of feces, 1 ml of bacterial cultures, 5-10 mg of animal tissues, or 10-20 mg of plant tissues.
Economic
AquaGenomic is priced at $199/kit for 300 minipreps, that is $0.66/miniprep or 1/4 of the price of other genomic DNA isolation kits.
Comparison
Below is a comparison of AquaGenomic and a leading competitor. You can easily see the advantages and benefits of using AquaGenomic.
Competitor Q AquaGenomic
Format Solid support Aqueous Solution
Price ($/mini) 2.10-6.46 0.66
Chaotropic salt Yes No
Multiple kits Yes, mini, midi, maxi; animal, plant, blood, etc. No, one kit for all sizes and materials
# of solutions 6-7 1
Lysate for PCR No Yes
Scalable No Yes
Img14.png
Comparison of genomic DNA isolated from different mouse tissues by AquaGenomic. Approximately 10 mg of tissue or 100 ul of blood were processed using the AquaGenomic Kit. Final genomic DNA was suspended in 50 ul deionized water. Five-microliter of each prep was run in a 1% agarose gel.
Brief Protocol
(Please refer to the Instruction Manual for DNA extraction from bacteria, cultured cells, animal tissues, plant tissues, soil, saliva, blood, and DBS, etc.)
AquaGenomic Swab Protocol
Cotton swabs are commonly used in forensic evidence collection. However, existing DNA extraction methods can recover only ~100-500 ng of DNA from a dried specimen swab as the majority of DNA remains entrapped within the cotton matrix. With AquaGenomic, ~5-8 ug of DNA can be extracted from a dried specimen swab. It is possible to use cotton swabs for specimen collection, transportation, storage, and DNA extraction for low-complexity and low-cost biobanking, biosurveillance, and epidemiological research applications.
1. Collect the specimen. Use a cotton swab to soak up the specimen (~100-200 ul), such as blood, saliva, mucus, semen, feces, cultured mammalian or bacteria cells, homogenized animal or plant tissues, or any other potential sources of biospecimens. Air-dry the specimen swab at ambient temperature (20-50 °C) for >24 hours. The dried specimen swabs can be shipped at ambient temperature and then stored at room temperature in sealed paper envelopes or plastic bags with desiccant for many years.
2. Extract the proteins and small molecules (dried blood swabs). Cut off the specimen swab tip into a 1.5-ml microfuge tube. Add 400 ul deionized water and soak the swab for >30 min. Use a 1-ml pipet tip to smash the swab ~10 times and press it to the bottom of the tube to squeeze out the solution. Transfer as much liquid as possible (~300 ul) to a new 1.5-ml microfuge tube for analysis of plasma proteins or small molecules.
3. Extract the DNA. Add 300 ul AquaGenomic to the 1.5-ml tube containing the wet swab from Step 2 or a new dried specimen swab. Incubate at 75-85 °C for 30 min. Centrifuge briefly to collect any condensation. Use a 1-ml blue pipet tip to smash the swab ~10 times and press it to the bottom of the tube to squeeze out the solution. Alternatively, homogenize the samples in AquaGenomic in 0.5-ml screw capped tubes with a multichannel bead beater. Transfer as much liquid as possible (~200 ul) to a new 0.5-ml microfuge tube or use a microfuge spin bucket to recover all the lysate from the swab to maximize the DNA yield (Optional: To use the crude lysate for PCR, dilute an aliquot of the crude lysate with 10-20 vol of deionized water and use 0.25 ul of the diluted lysate in a 25-ul PCR reaction).
4. Purify the DNA. Centrifuge at 10,000 xg for 5 min to pellet any debris in the crude lysate. Transfer the clear lysate (~200 ul) to a 0.5-ml tube and mix with 1 vol of isopropanol (~200 ul). Centrifuge at 10,000 xg for 5 min to pellet the DNA. Flip the tube to discard the isopropanol supernatant as completely as possible. Gently shoot 50% isopropanol from a squirt bottle to fill up the tube. Flip the tube to discard the isopropanol rinse as completely as possible. Tap and place the tube upside down on a clean paper towel to remove residual solution. Air-dry the DNA pellet for 5-10 min. Add 100 ul of deionized water to the DNA pellet and let it rehydrate for >15 min. Vortex or pipet up and down to solubilize the DNA. Centrifuge to pellet any insoluble material and transfer the clear DNA solution to a new tube.
.
Ordering Information
Product Name: AquaGenomicTM Kit
Product Number: 2001, 2030
Application: Isolation of genomic DNA from cells and tissues. For in vitro research use only.
Size: The kit is sufficient for the preparation of
2001: 10 minipreps (use 100 ul AquaGenomic per 1 million culture cells)
2030: 300 minipreps
Kit Contents: The AquaGenomic Kit includes the following items
2001: 1 ml AquaGenomic Solution, User Manual
2030: 30 ml AquaGenomic Solution, User Manual
Price:
2001: $10 each
2030: $199 each
Ordering: To order, please click the "BUY" button below.
AQUABLUER
AquaBluerTM is a proprietary redox indicator. Viable cells turn AquaBluer from its oxidized form (nonfluorescent, blue) to the reduced form (fluorescent, red). The fluorescence intensity of AquaBluer at 540ex/590em is proportional to the number of viable cells in the sample. It can be used to assess cell viability, cell proliferation, and cytotoxicity. It is nontoxic, simple to use, sensitive, reproducible, and has a broad assay range. Most importantly, it reduces your assay cost 3 folds over Alamar Blue and 26 folds over MTT.
- Features
- Comparison
- Ordering Information
- Brief Protocol
- Instruction Manual
- FAQ
Features
Colorimetric and fluorescent redox indicator for cell viability assay
Perform viability assay with a fluorescence plate reader or absorbance plate reader.
Simple to use and assay is performed in culture medium
Just add AquaBluer to the culture, incubate and read. No PBS, no SDS and no DMSO.
Nontoxic, sensitive, reproducible, and with a broad assay range
Not a terminal assay. AquaBluer-treated cells may still be used in other analyses.
Costs 3X less than Alamar Blue assay and 26X less than MTT assay
More saving means more research to you.
Comparison of Alamar Blue, AquaBluer and MTT Assays
| Alamar Blue | AquaBluer | MTT |
Detection mechanism | Redox indicator | Redox indicator | Redox indicator |
Protocol | 1. Replace culture medium with PBS-based Alamar Blue. 3. Read. | 1. Replace culture medium with medium-diluted AquaBluer or just add undiluted AquaBluer to the culture. 2. Incubate for 4 hrs (Your cells remain in their accustomed culture medium.). 3. Read | 1. Replace culture medium with medium-diluted MTT. 2. Incubate for 4 hrs. 3. Add SDS-HCl and pipet to lyse the cells. 4. Incubate for 4 hrs to solubilize the formazan dye. 5. Pipet to mix thoroughly and read. |
Price per kit | $400 /100 ml | $199 /15 ml | $340 /10 ml |
# of assays per kit | 10,000 | 15,000 | 1,000 |
Assay range | 600-75,000 cells | 600-150,000 cells | 2,000-250,000 cells |
aquabluer_assay
AquaBluer has a wider dynamic assay range than Alamar Blue
Brief Protocol
1. Set up a cytotoxicity assay
Seed the cells at 6000-8000 cells/100 ul/well in 96-well culture plates and let the cells grow overnight. Add serially diluted test compounds to each well. Include 4 “No-cells” and 4 “No-drug” wells as 0% and 100% viability controls. Return the plate to the CO2 incubator for 48-72 hrs.
2. Start AquaBluer assay
Add 100 ul AquaBluer solution (15 ml in the kit for 15,000 assays) to 10 ml culture medium in a dispensing reservoir. Pipette up and down to mix well. Replace the culture medium in each well with the 100X diluted AquaBluer medium. Alternatively, directly add 1 ul of undiluted AquaBluer solution to each 100 ul well, convenient for viability assay of fresh suspension cultures. Return the plate to the CO2 incubator for 4 hrs.
3. Raw data acquisition
Remove the plate from the incubator. Place the plate in a fluorescence plate reader and read the fluorescence intensity at 540ex/590em.
4. Viability and IC50 calculation
Subtract the average of fluorescence value (RFU) of the No-cell control (background) from all other RFU values. Convert the test RFU values to % viability using the formula:
% Viability = (RFUtest / RFUveh) x 100,
where RFUveh is the average RFU of the No-drug wells. Enter the % viability values and corresponding log test compound concentrations into a non-linear regression program such as Prism and use the Four Parameter Model to obtain the IC50 values and dose-response curve of the test compounds.
Ordering Information
Product Name: AquaBluerTM Kit
Product Number: 6001, 6015
Application: Assessing cell viability, cell proliferation, and cytotoxicity.
Size: The kit is sufficient for performing
6001: 1,000 assays in 96-well format using 1 ul AquaBluer in 100 ul medium
6015: 15,000 assays
Kit Contents: The AquaBluer Kit includes the following items
6001: 1 ml AquaBluer solution, Instruction Manual
6015: 15 ml AquaBluer solution, Instruction Manual
Price:
6001: $10 each
6015: $199 each
Ordering: To order, please click the "BUY" button below.
AquaPlasmid
is a multifunctional reagent for plasmid DNA purification without using columns. This single solution completes cell suspension, cell lysis, plasmid DNA extraction and cell debris removal in a single tube. AquaPlasmid is nontoxic. It does not contain hazardous chemicals, such as guanidine hydrochloride, used in column-based methods. The multifunctionality makes AquaPlasmid the most economic ($0.5/miniprep) and environment-friendly (nontoxic) plasmid purification product on the market. The isolated plasmid DNA is pure and suitable for all downstream applications, including automated DNA sequencing.
Features
Comparison
Ordering Information
Brief Protocol
Instruction Manual
FAQ
Features
Innovative
AquaPlasmid extracts plasmid DNA without using alkaline lysis and without denaturing the DNA.
Simple
AquaPlasmid completes cell suspension, cell lysis, plasmid extraction and debris removal in a single tube.
Scalable
A small 60-ml AquaPlasmid kit can be used for 300 mini, 30 midi, or 3 maxi preps.
Nontoxic
No phenol, no chloroform, and no guanidine salts.
Lowest cost
Only $0.50/miniprep, 1/3 of column-based purification kits.
Sequencing
AquaPlasmid purified plasmid DNA is pure (A260/A280 = 1.8-1.9) and suitable for routine molecular biology applications, including automated DNA sequencing.
Comparison
Comparison of AquaPlasmid and column-based plasmid purification
Column-based AquaPlasmid
Cell lysis Alkaline lysis Non-alkaline lysis
Isolation mechanism
Selective binding
Selective extraction
Isolation format Solid support
Aqueous solution
Scalable
No
Yes
# of Solutions
7 1
Use toxic chemicals
Guanidine salts * None
Cost ($/miniprep) 1.50 0.50
* Headaches in the lab? Column-based plasmid purification kits use large amount of buffers (e.g., Buffer N3 and PB) containing 5 M guanidine salts, which are not biodegradable. As minipreps are the "drinks" and "foods" of a molecular biology lab, guanidine salts are likely everywhere in your centrifuges, racks, counters, floors, sinks and sewage. When you rinse the culture flasks with Bleach (sodium hypochlorite) into the sinks, mop the floors or wipe the benches with Bleach-containing cleaning solutions, toxic and mutagenic gases, such as hydrogen cyanide, hydrogen chloride and reactive chloramines, etc., will be released into the lab, which may cause headache and other symptoms.
* Deformities in 20-40% of frogs around the cities? 10-20 times more frogs around major cities are found to have deformed limbs. When buffers containing guanidine salts are dumped into the sewage (hundreds of tons a year!), they could react with Bleach from household and industrial washers. The toxic reaction products could contaminate the water and harm the aquatic lives.
Brief Protocol
1. Harvest the Cells
Transfer 1.5 ml of overnight bacterial culture to a 1.7-ml microfuge tube. Centrifuge at 14,000 xg for 1 min at 22 °C to pellet the bacteria. Aspirate or flip the tube forcefully a few times to remove the culture medium as completely as possible.
2. Lyse the Cells
Add 200 ul of AquaPlasmid solution to the cell pellet. Immediately touch-vortex (a few seconds on and a few seconds off) at top speed for 10-20 sec to fully suspend the cells. Incubate at 22 °C for 10 min to lyse the cells.
3. Remove the Debris
Incubate the crude lysate at -20 °C for 5 min or on ice for 10 min to induce precipitation. Centrifuge at 14,000 xg for 5-10 min at 22 °C to pellet the cellular debris. IMPORTANT: Do not invert or vortex or shake the crude lysate after the lysis incubation or it will cause genomic DNA contamination (see the figure below)!
AquaPlasmid QC
4. Pellet the Plasmid DNA
Transfer the clear lysate (~180 ul, be careful not to transfer any cellular debris) to a clean 0.5-ml microfuge tube. Add 0.5 vol (90 ul) of isopropanol to the lysate (do not use more than 0.7 vol of isopropanol for DNA precipitation to avoid contamination). Touch-vortex at top-speed 5-10 times to mix well. Centrifuge at 14,000 xg for 5 min at 22 °C to pellet the plasmid DNA. Flip the tube (you may hold 6-10 tubes between your thumb and index finger at the same time) forcefully a few times to discard the supernatant.
5. Rinse the Plasmid DNA
To rinse the DNA pellet (nearly invisible), overflow the tube with 70% ethanol from a squirt bottle (be sure to rinse the inside of the lid as well), and then flip the tube with a swirling motion to discard the ethanol solution. Flip the tube forcefully a few times and blot it on a paper towel to remove residual ethanol. Leave the tube upside down on the paper towel for 5 min to air-dry the DNA pellet. Add 50 ul of deionized water (or TE buffer) to the DNA pellet and vortex for 30 sec to fully solubilize the DNA. Centrifuge at 14,000 xg for 2-3 min to pellet any insoluble (nearly invisible). Optional: transfer the DNA solution to a new tube.
Ordering Information
Product Name: AquaPlasmidTM Kit
Product Number: 1001, 1015, 1030, 1060
Application: Isolation of plasmid DNA. For in vitro research use only.
Size: The kit is sufficient for the preparation of
1001: 5 minipreps
1015: 75 minipreps
1030: 150 minipreps, or 15 midipreps, or 1.5 maxipreps
1060: 300 minipreps, or 30 midipreps, or 3 maxipreps
Kit Contents: The AquaPlasmid Kit includes the following items
1001: 1 ml AquaPlasmid solution, User Manual
1015: 15 ml AquaPlasmid solution, User Manual
1030: 30 ml AquaPlasmid solution, User Manual
1060: 60 ml AquaPlasmid solution, User Manual
Price:
1001: $10 each
1015: $59 each
1030: $99 each
1060: $149 each
Ordering: To order, please click the "BUY" button below.
AQUAPRESERVE
AquaPreserveTM is a multifunctional 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 extracts total DNA/RNA/proteins from whole blood; it recovers both cellular and cell-free circulating DNA/RNA/proteins in whole blood samples, therefore, maximizing the scientific value and utilities of the biospecimens. AquaPreserve is the only reagent that can extract intact RNA from frozen whole blood samples collected in common anticoagulants.
Features
Comparison
Ordering Information
Brief Protocol
Instruction Manual
FAQ
Use of AquaPreserve for total blood DNA/RNA biobanking
aquapreserve.png
Comparison
Comparison of Techniques for Biospecimen Preservation
Formalin | R*later | AquaPreserve | |
Mechanism of Stabilization | Cross-linking | High salt dehydration | Enzyme inactivation |
Damage to the Specimen | Yes | No | No |
Inactivation of Pathogens | Yes | No | Yes |
Offensive Odor | Yes | No | No |
DNA, RNA, and Protein Extraction | No | No | Yes |
Features
Streamline biospecimen pre-analytical workflow
AquaPreserve not only preserves but also extracts DNA/RNA/proteins from the biospecimen. It can be used to streamline the entire pre-analytical workflow, from specimen collection (immediately arrests gene expression and stabilizes the DNA/RNA/proteins) to DNA/RNA/protein extraction (does not use any other DNA, RNA, and protein extraction kits). Therefore, it reduces biospecimen pre-analytical variability, prevents sample cross-contamination, increases detection specificity, and improves data reproducibility and reliability.
Safeguard biospecimens during their transportation and storage
AquaPreserve/ProSink-stabilized specimens may be stored at 4 and 22 °C for weeks without causing RNA degradation. AquaPreserve serves as a safeguard against unexpected cold-chain disasters during shipping and storage, and makes it feasible to repeatedly thaw the frozen the specimens for QC/QA and for distribution to researchers.
Maximize the scientific value of the biospecimens
Presently you probably only store cellular DNA extracted from fresh blood and you don't have a reliable method to extract RNA from frozen whole blood collected in common anticoagulants (not in specialized PAXgene or Tempus RNA tubes). With AquaPreserve, you not only can extract blood DNA but also blood RNA, not only cellular DNA/RNA/proteins but also cell-free circulating DNA/RNA/proteins, from not only fresh but also archived frozen whole blood. It allows you to do more researches and generate more data previously impossible from your precious specimens.
Reduce the overall cost of biobanking
AquaPreserve enables the freezing of leftover blood samples obtained from routine testing for later DNA/RNA/protein extraction, therefore, opens up an unlimited and inexpensive biospecimen resource. AquaPreserve-stabilized the blood biospecimens may be shipped and stored at 4 or 22 °C for years without causing DNA degradation, therefore, reducing the energy consumption. Additionally, AquaPreserve extracts DNA/RNA/proteins from the specimens without needing other DNA, RNA, and protein extraction kits.
Comparison
Comparison of Techniques for Biospecimen Preservation
Formalin
R*later
AquaPreserve
Mechanism of Stabilization
Cross-linking High salt dehydration Enzyme inactivation
Damage to the Specimen
Yes No
No
Inactivation of Pathogens
Yes
No
Yes
Offensive Odor
Yes
No
No
DNA, RNA, and Protein Extraction
No
No Yes
dnasei treated blood
Use the volume ratio of 1:1:0.5 (blood:AquaPreserve:ProSink) for other extraction scales
Micro | Mini | Midi | Maxi | |
Blood (ul) | 50 | 250 | 2,000 | 4,000 |
AquaPreserve (ul) | 50 | 250 | 2,000 | 4,000 |
ProSink (ul) | 25 | 125 | 1,000 | 2,000 |
Centrifuge tubes | 0.6-ml | 1.5-ml | 15-ml | 15-ml |
DNA yield (ug) | 2-3 | 12-15 | 100-130 | 200-250 |
RNA yield (ng) | 50 * | 250 | 2,000 | 4,000 |
Extractions per #8060 and #9030 | 1,200 | 240 | 30 | 15 |
Price per extraction ($) | 0.21 | 1.03 | 8.27 | 16.53 |
* This is the RNA yield from human blood. For mouse blood, RNA yield may be 2500 ng/50 ul blood.
Frozen human ACD blood was thawed in 1 vol of AquaPreserve. Aliquots of the AquaPreserve lysed blood were mixed with ProSink and stored at -80, 22, and 37°C for 7 days. DNA and RNA were recovered from the cleared lysate by isopropanol precipitation. One tenth of the total DNA/RNA from 200 ul of whole blood was digested with DNase I and electrophoresized in a 0.8% non-denaturing agarose gel.
Brief Protocol
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 or shake to mix well or until the frozen blood sample is completely thawed in AquaPreserve (Do not thaw the 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, incubated at 22 °C for 20 min to degrade the RNA, aliquoted and then mixed with AquaPreserve.).
2. Remove the proteins
Add 0.125 ml of ProSink (#9030) to the AquaPreserve lysed blood. Vortex for 1 min to mix well and incubate at 22 °C for >30 min (Blood DNA is now stable at 4-22 °C for a year, and blood RNA is now stable at -80 °C forever, 4 °C for 2 weeks, 22 °C for 7 days.). Centrifuge at 14,000xg for 10 min to pellet the proteins (save the pellet for protein recovery with ProMelt (#1115), if desired).
3. Pellet the DNA/RNA
Pour the supernatant (~0.7 ml) to a 1.5-ml microfuge tube preloaded with 0.8 vol (~0.56 ml) of isopropanol and vortex to mix. Centrifuge at 14,000 xg for 10 min to pellet the DNA/RNA. Decant to discard the supernatant (or save it for small molecule analysis) and rinse the DNA/RNA pellet with 70% ethanol twice. Suspend the DNA/RNA pellet in 50 ul of TE buffer or deionized water.
Use the volume ratio of 1:1:0.5 (blood:AquaPreserve:ProSink) for other extraction scales
Micro Mini Midi Maxi
Blood (ul) 50 250 2,000 4,000
AquaPreserve (ul) 50 250 2,000 4,000
ProSink (ul) 25 125 1,000 2,000
Centrifuge tubes 0.6-ml 1.5-ml 15-ml 15-ml
DNA yield (ug) 2-3 12-15 100-130 200-250
RNA yield (ng) 50 * 250 2,000 4,000
Extractions per #8060 and #9030 1,200 240 30 15
Price per extraction ($) 0.21 1.03 8.27 16.53
* This is the RNA yield from human blood. For mouse blood, RNA yield may be 2500 ng/50 ul blood.
AquaPreserve Buffy Coat DNA/RNA/Protein Extraction Protocol
If you need to recover the plasma for other assays or prepare DNA/RNA/proteins from large volume of blood while reducing the consumption of the extraction reagents, you may prepare buffy coat from whole blood or use existing frozen buffy coat samples for DNA/RNA/protein extraction. The protocol below is for processing ~2 ml of whole blood to obtain ~200 ul of buffy coat. If you need to process larger volume of whole blood in the original vacutainer (5-10 ml), you will simply scale up the reagent volumes proportionally.
1. Prepare the buffy coat
Centrifuge 2 ml of anticoagulated whole blood at 300 xg for 10 min at room temperature. Remove some plasma (~0.6-0.7 ml) without disturbing the buffy coat. Set the pipette at 100-ul and carefully suck up the grayish buffy coat while slowly moving the tip across the interface and taking up as little RBC as possible. Transfer the buffy coat to a 1.5-ml microfuge tube. Repeat it by taking 100 ul of plasma just above the interface. The total volume of buffy coat recovered is about 1/10 of the blood volume, that is, ~200 ul.
2. Lyse the blood cells
Add one volume (~200 ul) of AquaPreserve (don’t forget to invert the reagent bottle to mix the reagent well before dispensing) to the buffy coat. Vortex to mix well.
3. Recover the DNA/RNA
Add 0.8 volume (~320 ul) of isopropanol to the cell lysate. Vortex to mix well. Centrifuge at 12,000 xg for 5 min at room temperature to pellet the DNA/RNA. Transfer 0.4 ml protein-containing supernatant to a 2-ml tube for protein recovery. Remove the remaining supernatant from the DNA/RNA pellet as much as possible. Fill up the microfuge tube with 50% isopropanol to rinse the DNA/RNA pellet and quickly decant to discard the isopropanol solution. Repeat the isopropanol rinse once. Tap the tube on a clear paper towel to remove residual isopropanol and leave the tube up side down to air dry the DNA/RNA pellet for 5-10 min. Add 100 ul of deionized water to the pellet, and vortex to suspend the DNA/RNA pellet. Incubate at room temperature for 5-10 min and centrifuge again to pellet any insoluble. Transfer the DNA/RNA solution to a new tube and store at –20 °C.
4. Recover the proteins
Add 4 volume (1.6 ml) of acetone to the isopropanol supernatant obtained after DNA/RNA precipitation. Shake or vortex to mix well. Centrifuge at 12,000 xg for 5 min to pellet the proteins. Decant to discard the supernatant. Immediately add 100 ul of ProMelt (#1150, order separately) to the wet protein pellet. Pipet and vortex to solubilize the proteins. Centrifuge to pellet any insoluble. The protein solution may be loaded directly to SDS-PAGE.
(Note: If the buffy coat contains large amount of RBC, you may need to use 2 volumes of AquaPreserve for the extraction or try various volume of ProSink (#9030) for protein precipitation to reduce hemoglobin contamination of the DNA/RNA pellet.)
Ordering Information
Product Name: AquaPreserve Kit
Product Number: 8001, 8060
Application: Biospecimen preservation and extraction. For in vitro research use only.
Size: The kit is sufficient for the preparation of
8001: 4 DNA/RNA minipreps (use 0.25 ml AquaPreserve for 0.25 ml blood)
8060: 240 DNA/RNA minipreps
Kit Contents: The AquaPreserve Kit includes the following items
8001: 1 ml AquaPreserve Solution, Instruction Manual
8060: 60 ml AquaPreserve Solution, Instruction Manual
Price:
8001: $10 each
8060: $199 each
Ordering: To order, please click the "BUY" button below (order ProSink and ProMelt separately).
AQUARNA
AquaRNATM is a multifunctional aqueous solution-based reagent for DNA, RNA, and protein extraction. This single solution will lyse the cells, inactivate degradative enzymes, and extract DNA, RNA, and proteins. DNA and RNA are recovered from the cell lysate by isopropanol precipitation, while proteins remain soluble in the isopropanol solution and can be recovered by acetone precipitation. AquaRNA enables concurrent isolation of DNA, RNA, and proteins from the same specimen without using different DNA, RNA, and protein extraction kits.
Features
Comparison
Ordering Information
Brief Protocol
Instruction Manual
FAQ
ar11.png
Features
RNA myths
AquaRNA will change the way you think and work with RNA by simply inactivating and removing endogenous contaminating RNases in your RNA samples.
"RNA work must be done at designated RNA-only workbench, use RNA-only equipment and RNase-free consumables." - Unnecessary. If you won't touch the inside of your RNA tube, your chance of getting RNA degradation by using regular reagents and tubes is slim to none.
"I am always anxious until I see the results of my RNA experiments." - If the RNA sample is not contaminated with RNases, you will get consistent and reproducible results every time.
RNase Decontamination
AquaRNA may be used to remove contaminating RNases from purified RNA or remove contaminating RNases in purified plasmid DNA for in vitro transcription.
Not only for RNA
Comparison
| Competitor | AquaRNA |
Isolation Format | Solid support | Aqueous solution |
Price ($/mini) | 3.84 | 0.66 |
Mercaptoethanol | Yes | No |
Scalable | No | Yes |
# of Solutions | 4 | 1 |
Small RNAs | Not recovered | Recovered |
DNA | Not recovered | Recovered |
AquaRNA can extract DNA, RNA, and proteins from the same biological samples concurrently and allow you to maximize the value of your biospecimens.
Simple
Due to the multifunctionality of the AquaRNA solution, its protocol is very simple. After cell lysis, DNA/RNA are precipitated with isopropanol and proteins are precipitated with acetone. No columns are needed.
No phenol and chloroform
AquaRNA extracts DNA/RNA/proteins without using phenol and chloroform. You will not have problem solubilizing the protein pellet as you often encounter using organic extraction.
Scalable
One AquaRNA Kit does all. No need to purchase separate mini, midi, maxi, and HTP kits.
Comparison
Competitor
AquaRNA
Isolation Format
Solid support
Aqueous solution
Price ($/mini)
3.84
0.66
Mercaptoethanol
Yes
No
Scalable
No
Yes
# of Solutions
4
1
Small RNAs
Not recovered
Recovered
DNA
Not recovered
Recovered
bioanalyzer.jpg
Total RNA was extracted from an E. coli culture and the RNA integrity was measured with Agilent 2100 Bioanalyzer.
rna gel for web3.jpg
DNA/RNA was extracted from bacterial culture, mammalian culture, and rat liver tissue with AquaRNA, and treated with or without DNase I. Shown are the DNA, 28S (23S), 18S (16S), and 5S RNA bands.
Brief Protocol
(Please refer to the Instruction Manual for DNA/RNA/protein extraction from bacteria, cultured cells, animal tissues, and plant tissues, etc.)
1. Harvest the Cells
Pellet ~0.5-2 million cultured cells in a 1.5-ml microfuge tube by centrifugation at 14,000 xg for 60 sec. Aspirate or decant to discard the supernatant. (Note: Centrifuging to pellet the cells may not be needed as long as 1 vol of AquaRNA is mixed with 1 vol of cell suspension for DNA/RNA extraction.)
2. Extract the DNA/RNA
Add 100 ul AquaRNA solution (30 ml in the kit for 300 minipreps) to the cell pellet (or equal volume of cell suspension). Vortex vigorously for 60 sec to lyse the cells.
3. Pellet the DNA/RNA
Add 0.8 vol (~100 ul) of isopropanol to the crude lysate and vortex to mix. Centrifuge at 14,000 xg for 5 min to pellet the DNA/RNA. Decant to discard the supernatant (Note: Proteins remain in the isopropanol supernatant and can be recovered by precipitation in 4 vol of acetone.) and rinse the DNA/RNA pellet with 70% ethanol twice. Suspend the DNA/RNA pellet in 100 ul of TE buffer or deionized water. Centrifuge again to pellet any insoluble material and transfer the DNA/RNA solution to a new tube. Remove DNA in the DNA/RNA prep by DNase I digestion.
Ordering Information
Product Name: AquaRNATM Kit
Product Number: 5001, 5030
Application: Isolation of DNA/RNA/proteins. For in vitro research use only.
Size: The kit is sufficient for the preparation of
5001: 10 minipreps (use 100 ul AquaRNA per million culture cells)
5030: 300 minipreps
Kit Contents: The AquaRNA Kit includes the following items
5001: 1 ml AquaRNA Solution, Instruction Manual
5030: 30 ml AquaRNA Solution, Instruction Manual
Price:
5001: $10 each
5030: $199 each
Ordering: To order, please click the "BUY" button below.
Aquastool
Stool is an accessible and underutilized noninvasive source of biospecimen. Fecal DNA/RNA originated from the host, intestinal bacteria, viruses, fungi, or parasites, and incompletely digested foods are molecular fingerprints of the host and its health. AquaStoolTM is a multifunctional aqueous solution-based reagent for fecal sample stabilization, DNA and RNA extraction and PCR inhibitor removal. It may be used to extract fecal DNA for non-invasive genotyping of transgenic animals. Fecal sampling is not limited by animal age, physiopathological condition, or sampling frequency. AquaStool may also be used to preserve and extract DNA/RNA from human stools for host and gut microbiota research.
Features
Comparison
Ordering Information
Brief Protocol
Instruction Manual
FAQ
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Features
Multi-functionalities
AquaStool combines the functions of specimen stabilization, DNA/RNA extraction, and PCR inhibitor removal in a single solution.
Non-invasive genotyping
AquaStool can be used to extract fecal DNA and enables noninvasive genotyping of transgenic animals and monitoring of wildlife animals.
Recover host and microbial DNA and RNA in feces
AquaStool extracts fecal DNA and RNA originated from the host, commensal bacteria, invasive viruses, fungi, or parasites, and incompletely digested foods, enabling the study of human and animal microbiome; diagnosis of bacterial, viral, fungal, and parasitic infections.
Powerful lysis
The powerful combination of AquaStool, sand and sonication lysis can produce 100-200 ug of DNA from 50 mg of feces. The sheared DNA is ready for next generation sequencing and could reveal a different but "true" microbiota profile of your samples.
Not only for extraction but also for specimen stabilization
AquaStool may be used to collect fecal specimens in the field. It will stabilize the DNA/RNA and allow the shipping of the fecal samples to the laboratory at ambient temperature, therefore, streamline fecal sample collection, transport and DNA/RNA extraction with a single reagent.
Comparison of tail DNA and fecal DNA extraction
Tail DNA | Fecal DNA | |
Reagent | AquaGenomic * | AquaStool |
Specimen | Tail or ear snip | Fecal pellet |
Invasive | Yes | No |
Repeated sampling | Not allowed | Allowed |
Age limitation | Under 21 days | Unlimited |
Use lysate directly for PCR | Yes | No |
Cost per extraction | $0.33 | $1.00 |
GI microbial DNA | No | Yes |
fDNA-RNA
1 2 3 4
Figure 1. AquaStool purified fecal DNA and RNA. Aliquots (5 μl) of the extracted DNA and RNA were electrophoresized in a native 0.8% agarose gel.
DNA ladder.
DNA extracted by vortexing.
DNA/RNA extracted by sonication (The sheared DNA is ready for next generation sequencing. RNA contamination will not affect sequencing).
DNase I treated RNA.
Comparison
Comparison of tail DNA and fecal DNA extraction
Tail DNA | Fecal DNA | |
Reagent | AquaGenomic * | AquaStool |
Specimen | Tail or ear snip | Fecal pellet |
Invasive | Yes | No |
Repeated sampling | Not allowed | Allowed |
Age limitation | Under 21 days | Unlimited |
Use lysate directly for PCR | Yes | No |
Cost per extraction | $0.33 | $1.00 |
GI microbial DNA | No | Yes |
We offer AquaStool for fecal DNA extraction and AquaGenomic for tail DNA extraction. Here is a comparison of the two methods.
Comparison of tail DNA and fecal DNA extraction
Tail DNA
Fecal DNA
Reagent AquaGenomic * AquaStool
Specimen
Tail or ear snip
Fecal pellet
Invasive
Yes
No
Repeated sampling
Not allowed
Allowed
Age limitation
Under 21 days
Unlimited
Use lysate directly for PCR
Yes
No
Cost per extraction
$0.33 $1.00
GI microbial DNA
No
Yes
* AquaGenomic (#2030) may also be used to extract fecal DNA from animal feces, where fecal PCR inhibitors are removed with the AquaPrecipi solution (#3015). If you are interested in mitochondrial DNA typing, we recommend the use of AquaGenomic for fecal DNA extraction.
Brief Protocol
(Please refer to the Instruction Manual for fecal DNA/RNA extraction from human stool specimens.)
AquaStool mouse fecal DNA extraction protocol
1. Collect the fecal sample
Transfer a mouse to a cage floored with a clean paper towel. Scoop up a fecal pellet with a microfuge tube labeled with the animal ID (fecal pellets may be stored at RT for months after being air-dried by incubating in open tubes on a 37 °C heat bloc for 24 hrs).
2. Extract the DNA
Add 150 ul AquaStool (30 ml in the kit for 200 extractions) to each fecal pellet. Soak the pellet until it becomes soften and vortex vigorously to homogenize the fecal material (if needed, use a pipette tip to break up the fecal pellet). For microbial DNA extraction, vortex the fecal material in the presence of white sand or homogenize it with a bead beater.
3. Remove the Debris
Centrifuge the sample at 14,000 xg for 10 min to pellet the debris.
4. Pellet the DNA
Transfer the supernatant (~90 ul) to a new 0.5-ml microfuge tube. Add 0.8 vol (~72 ul) of isopropanol and vortex to mix. Centrifuge at 14,000 xg for 5 min to pellet the DNA. Decant to discard the supernatant and rinse the DNA pellet with 70% ethanol twice. Suspend the DNA pellet in 100 ul of TE buffer or deionized water. Centrifuge again to pellet any insoluble material and transfer the DNA solution to a new tube (Note: It is critical to centrifuge the DNA solution again before adding it to a PCR reaction, as some insoluble material, which contain PCR inhibitors, may develop during storage.).
AquaGenomic mouse fecal DNA extraction protocol
1. Collect the fecal pellet
Transfer a mouse to a cage floored with a clean paper towel. Scoop up a fecal pellet with a microfuge tube labeled with the animal ID (fecal pellets may be stored at RT for months after being air-dried by incubating in open tubes on a 37 °C heat bloc for 24 hrs).
2. Extract the DNA
Add 200 ul of AquaGenomic solution to each fecal pellet. Incubate at 85 °C for 20 min (For mitochondrial DNA extraction, add Proteinase K to the AquaGenomic solution to 100 ug/ml, incubate the sample at 60 °C for 2 hrs and then at 95 °C for 10 minutes to inactivate the Proteinase K). Homogenize the heat-treated sample by vortexing or pipetting or bead-beating with a multichannel bead-beater.
3. Pellet the Debris
Centrifuge at 12,000 xg for 4 min to pellet the debris. Transfer the supernatant (~100 ul) to a new 0.5-ml microfuge tube.
4. Pellet the DNA
Add 0.5 vol (~50 ul) of AquaPrecipi (#3015, order separately) and 0.5 vol (~50 ul) of 95-100% of ethanol. Vortex for 60 sec and centrifuge at 12,000 xg for 5 min to pellet the DNA. Decant to discard the supernatant. Fill the tube with 70% ethanol from a squirt bottle, then flip the tube to discard the ethanol solution. Repeat the 70% ethanol rinse 2 times. Place the tube upside down on a clean paper towel for 5-10 min to air-dry the DNA pellet. Add 100 ul of TE buffer or deionized water to the DNA pellet, pipette or vortex vigorously to suspend the DNA. Centrifuge at 12,000 xg for 10 min to pellet any insoluble material, which contains residual PCR inhibitors, and transfer the clear DNA solution to a new tube.
.
Ordering Information
Product Name: AquaStool Kit
Product Number: 7001, 7030
Application: DNA/RNA extraction from fecal specimens. For in vitro research use only.
Size: The kit is sufficient for the preparation of
7001: 6 extractions (use 150 ul AquaStool for a mouse fecal pellet)
7030: 200 extractions
Kit Contents: The AquaStool Kit includes the following items
7001: 1 ml AquaStool Solution, Instruction Manual
7030: 30 ml AquaStool Solution, Instruction Manual
Price:
7001: $10 each
7030: $199 each
Ordering: To order, please click the "BUY" button below.
BLOOD BIOBANKING
Blood to DNA RNA
- AquaPreserve for streamlining total blood DNA/RNA biobanking
- AquaPreserve for maximizing the value of blood biospecimens
- AquaGenomic for streamlining total blood DNA biobanking
- AquaGenomic enables low-cost dry blood swab biobanking
Blood is the most collected biospecimen in routine medical care and clinical trials. Millions of archived blood samples are currently stored in biorepositories around the world. These blood samples are vital to medical research (e.g., understand the pathogenesis), drug development (identify and validate drug targets), diagnostic test development (discover disease biomarkers), and personalized medicine (predict patient's response to treatments). We have developed two aqueous solution based reagents, AquaGenomic and AquaPreserve, for total blood DNA and total blood DNA/RNA preservation and extraction from either fresh or frozen whole blood specimens. They may be used to streamline the workflow of blood biobanking and maximize the scientific value of blood biospecimens.
AquaPreserve for streamlining total blood DNA/RNA biobanking
There are several major challenges in translational research related to biobanking. One is the availability of clinically annotated biospecimens. The standard method of obtaining specimens through clinical trials not only is costly but also may take years or decades to meet the enrollment target, causing the delay or abandoning of many clinical studies. On the other hand, millions of leftover blood samples from routine medical tests are discarded everyday. If these blood specimens, which are linked to patient's electronic medical records (EMR) and represent numerous diseases, could be cryopreserved for later DNA/RNA/protein extraction and analysis, they would become an unlimited biospecimen resource to basic and clinic investigators. The second challenge is the quality of the biospecimens. If a specimen was not immediately "frozen" and kept at a "freezing" state until analysis, an observed molecular change, such as gene expression profile, may merely be the result of ex vivo alternation in the specimen. Fortunately, for most qualitative or mutational studies, specimens that are not instantly arrested, such as blood samples leftover from routine testing, remain adequate for PCR detection and sequencing analysis. The third challenge is the variability in biospecimen handling. Different biobanks and laboratories may use different methods for specimen collection, transport, storage, and DNA/RNA extraction. These differences may artificially change the quantity and quality of the recovered analytes, or introduce contaminants, which may lead to irreproducible, or incomparable, or even misleading results and conclusions.
AquaPreserve was developed to meet these challenges. First, AquaPreserve can be used to extract DNA/RNA/proteins from frozen whole blood collected in common anticoagulants, so it is now technically possible to freeze and save leftover blood samples from routine tests as an unlimited biospecimen resource for later DNA/RNA/protein extraction and analysis. Secondly, AquaPreserve can be used to stabilize blood DNA/RNA/proteins at blood collection and prevent continued change of these biomolecules during specimen shipping, storage, and extraction. And thirdly, because AquaPreserve combines preservation with extraction, it can be used to streamline the entire biospecimen workflow from collection to extraction, and thus reduce pre-analytical variability and prevent cross-contamination with unrelated DNA/RNA/proteins.
Below is an outline of how to use AquaPreserve for blood DNA/RNA/protein biobanking.
1. Donors
The laws and regulations governing human subject protection and privacy must be strictly adhered to. Informed consent must be obtained from all donors. Patient selection should represent a broad range of human diseases but focus may be placed on diseases with unmet need, having suspected genetic etiology, and patients with well annotated EMRs. All donors must be de-identified.
2. Collection
Blood specimens may be acquired by two ways. One is to obtain excess blood samples after the prescribed clinical tests have been completed. These blood samples are usually stored at 4 °C and available in 5-7 days after the blood draw. They are suitable for blood DNA extraction and analysis. They may also be used for blood RNA extraction for traditional cDNA sequencing and next-generation sequencing. However, for gene expression profiling, the possibility of altered expression of target genes prior to RNA extraction must be investigated. Another way is to take an extra tube of blood from the patient at the time of the scheduled blood draw. The blood samples can be mixed with one blood volume of AquaPreserve immediately following blood draw and then with 0.5 blood volume of ProSink to stabilize the blood RNA. They are suitable for all types of DNA and RNA analyses.
3. Transport
The AquaPreserve stabilized blood samples will be barcoded, linked to their de-identified donors' EMRs, and transported to the biobank overnight in wet ice.
4. Storage
Upon arriving at the biobank, the AquaPreserve/ProSink stabilized blood samples may be stored at 22 °C over night or 4 °C for 2 weeks or at -80 °C indefinitely before DNA/RNA extraction (Note: Do not store AquaPreserve/ProSink stabilized blood samples at -20 °C because it reduces RNA yield significantly by unknown mechanism). Unlike frozen plain blood, AquaPreserve/ProSink stabilized blood samples can tolerate freezer temperature fluctuation or sample thawing due to the opening of the freezer, temporarily removing the samples to room temperature, or in case of power outage and freezer breakdown.
5. Distribution
AquaPreserve/ProSink stabilized blood samples can be thawed and aliquoted for QC/QA testing or distribution to other investigators. In contrast, freeze-and-thawing is strictly prohibited for frozen plain blood as blood RNA will be degraded by freeze-and-thawing.
6. Extraction
First, the stabilized blood sample is centrifuged to pellet the proteins, and then total blood DNA/RNA is precipitated from the clear lysate with one lysate volume of isopropanol. The extraction does not require plasma removal, RBC lysis, Proteinase K digestion, phenol chloroform extraction, or spin column purification, which are commonly employed in other blood DNA and RNA extraction methods. Because the entire specimen processing is streamlined with AquaPreserve, pre-analytical variables are reduced or eliminated.
Comparison of P'Xgene and AquaPreserve for blood DNA and RNA extraction
P'Xgene
AquaPreserve *
Yield of RNA from 2.5 ml blood 2.5-5 ug 2.5-5 ug
Cost of RNA from 2.5 ml blood
$11/RNA vacutainer
+ $9/RNA kit
=$20 $0.45/standard vacutainer
+$8.29/AquaPreserve
+$0.77/ProSink
=$9.51
Yield of DNA from 2.5 ml blood
40-150 ug
100-150 ug
Cost of DNA from 2.5 ml blood
$1/DNA vacutainer
+$11/DNA kit
=$12
$0
# of centrifugation steps per extraction 11 (RNA) + 4 (DNA) = 15 3
Recovery of plasma DNA/RNA/proteins
No
Yes
* You may prepare DNA/RNA/proteins from larger volume of blood samples with 10x less reagents by extracting DNA/RNA/proteins from buffy coat, if buffy coat preparation would not alter the DNA/RNA/proteins of your interest. In other words, it may cost you <$1 to prepare DNA/RNA/proteins from a buffy coat prepared from 2.5 ml of whole blood using AquaPreserve.
AquaPreserve for maximizing the value of blood biospecimens
The value of a blood biospecimen relies on our ability to preserve and extract its analytes, such as DNA, RNA, and proteins, for downstream analyses. However, several current blood biobanking practices inadvertently reduce the scientific value and limit the utilities of blood biospecimens.
First, most blood samples are collected in regular anticoagulants not in specialized RNA stabilizing vacutainers, such as PAXgene and Tempus tubes, they must be processed timely (e.g., immediately for gene expression profiling, or within 48 hours for cDNA sequencing). Due to logistic and technical difficulties, most biorepositories can only extract and store blood DNA but not blood RNA for analysis.
Secondly, although millions of frozen whole blood specimens originally obtained from routine care, clinical trials, and epidemiological studies are stored in biobanks around the world, there is no reliable method to extract RNA from these archived frozen blood specimens. Researchers can only use these archived frozen blood specimens for DNA but not RNA analysis.
Thirdly, the standard method for blood DNA and RNA extraction is to lyse the RBC with a hypotonic solution, remove the plasma and the released hemoglobin, and then extract the DNA and RNA from the enriched WBC using different DNA and RNA extraction kits. Even for blood samples collected in PAXgene and Tempus tubes, a centrifugation and washing step is required to remove the plasma and the released hemoglobin before extracting the cellular RNA from the WBC pellet. As a result, potential biomarkers in the plasma, such as viral DNA, cell-free circulating DNA, plasma microRNA, and proteins are all lost. Only cellular DNA and RNA are recovered for analysis.
AquaPreserve possesses three unique properties. First, it combines DNA/RNA/protein preservation with DNA/RNA/protein extraction. Second, it can extract total blood DNA/RNA/proteins from either fresh or frozen whole blood collected in common anticoagulants. And third, it can extract total blood DNA and RNA without removing the RBC and the plasma. With AquaPreserve, one may freeze down the blood samples immediately following the blood draw. There will be no need to immediately carry out DNA/RNA/protein extraction, as blood DNA/RNA/proteins can be extracted from the cryopreserved blood samples, including existing archived frozen blood samples, with AquaPreserve. Since removal of plasma and RBC is no longer required, both cellular and cell-free DNA/RNA/proteins can be recovered with AquaPreserve. The end result is that now you can extract not only blood DNA but also blood RNA, not only cellular DNA/RNA/proteins but also cell-free DNA/RNA/proteins from the precious blood specimens, and thus maximize the scientific value and utilities of the blood specimens.
The goal of biobanking is to preserve biospecimens for the study of diseases and disease biomarkers. If we are going to spend millions of dollars to extract and store cellular DNA, might as well recover and store both cellular and plasma DNA/RNA/proteins for analysis, because it will enable epigenetic, transcriptomic, proteomic, and miRNA studies of non-hereditary, common diseases.
AquaGenomic for streamlining total blood DNA biobanking
If you are mainly interested in blood DNA extraction and analysis, AquaGenomic may be a better choice. AquaGenomic is nontoxic. Its blood lysate may be used directly for PCR amplification without further DNA purification. Most uniquely, AquaGenomic can extract 10 ug of DNA from a dry blood swab, which is 100x higher than other DNA extraction methods. Therefore, AquaGenomic enables the use of cotton swabs as a low-cost and low-complexity biobanking tool to collect and store millions of blood samples at room temperature. Compared to standard deep freezer biobanking, you will not incur huge initial equipment and facility costs, as well as ongoing maintenance and energy costs.
Below is an outline of how to use swabs and AquaGenomic to streamline blood specimen collection, stabilization, transport, storage, and DNA extraction.
1. Donors
The laws and regulations governing human subject protection and privacy must be strictly adhered to. Informed consent must be obtained from all donors. Patient selection should represent a broad range of human diseases but focus may be placed on diseases with unmet need, having suspected genetic etiology, and patients with well annotated EMRs. All donors must be de-identified.
2. Collection
Blood samples may be obtained freshly by venipuncture, finger or heel sticks or received as leftover samples from clinical laboratories. Approximately 100-200 ul of whole blood can be collected with a cotton swab. To stabilize the specimens, the blood swabs are air-dried at 20-50 °C for at least 24 hours.
3. Transport
The dried blood swabs may be shipped at ambient temperature to the laboratory in sealed paper envelopes.
4. Storage
Upon arriving at the biobank, the dried blood swabs can be stored at 22 °C.
5. Plasma protein extraction
To extract the plasma proteins from the dried blood swab for serological testing, the swab tip is cut off into a 1.5-ml microfuge tube and soaked in 0.4 ml of deionized water or buffer. The swab is smashed with a 1-ml pipet tip and squeezed to the bottom of the tube. The protein solution is then transferred to a new microfuge tube.
6. DNA extraction
To extract the DNA, 300 ul of AquaGenomic is added to the wet swab above (or a dried blood swab) in a 1.5-ml microfuge tube. The sample is incubated at 85 °C for 30 min. The swab is then smashed with a 1-ml pipet tip and squeezed to the bottom of the tube. The crude lysate is transferred to a new microfuge tube and centrifuged to pellet any debris. The clear lysate is transferred to another new microfuge tube, mixed with 1 volume of isopropanol, and centrifuged to pellet the DNA. The DNA pellet is rinsed with 50% isopropanol, air-dried, and solubilized in 100 ul of deionized water.
AquaGenomic enables low-cost dry blood swab biobanking
Most large-scale biobanks enroll volunteers from the general population. The donor sizes are less than one million. A million participants from the general population is not large enough to cover many diseases, especially rare diseases (1 in 2000-2500). In order to have a full representation of all diseases, rare and common, at different disease stages, under different treatment conditions and timelines, and in different age, gender, race, environment and other demographics, tens of millions of participants are needed.
How can we have that many participants and samples? Recruiting patients with residual blood samples may be a solution. There are approximately a billion blood draws a year in the US. If a portion of those leftover blood samples can be saved, we could have tens of millions of patient blood samples in a couple of years, potentially covering all diseases and health conditions as these blood samples are from patients not from the general population. And this currently wasted resource is unlimit. Furthermore, since multiple blood samples are often taken from the same patients before and after disease progression and before and after treatment, they are invaluable or even indispensable for dissecting and differentiating genetic and epigenetic changes caused by the diseases and/or by the treatments.
How can we handle that many samples? The current standard practice of immediately processing the blood samples, freezing the blood components, and extracting the genomic DNA not only is cost prohibitive but also logistically impractical for such a scale. A possible solution is to store the leftover blood samples as dry blood swabs at room temperature for decades. Dry blood swab is simple enough that most hospitals and clinical laboratories can do it. There will be no large initial investment in freezers, facilities, and man power. There will be no large ongoing maintenance and energy costs. You won’t even need to extract the DNA until the investigators have their projects in place, which is another large upfront saving to the biobanks. We don’t knows if all or which samples will be utilized in advance, why bother to waste the money to extract the DNA now? Furthermore, the dry blood swabs will preserve not only genomic DNA but also cell-free DNA, not only DNA but also proteins and small molecule analytes for unforeseen future applications and biomarker research. Aquagenomic's efficient and differential DNA and protein extraction from cotton swabs makes the creation of fully represented large blood biobanks using leftover blood samples from millions of patients possible [1].
The leftover blood sample approach may be particularly suited for recruiting pediatric patients, who may have less blood samples and do not want additional, unnecessary needling. They can benefit from the participation if the sequencing data will be added to their EHR for their future uses. After all, to enable precision medicine patients would need to have their sequencing data on file in their EHRs.
Some unique utilities of a pediatric dry blood swab biobank may include the following.
1. Discover disease-causing germline mutations, as pediatric specimens contain less somatic mutations induced by aging and the environment.
2. Serve as control for discovering somatic mutations that appear later in life and contribute to aging and adult onset diseases.
3. Use in longitudinal studies to compare genetic and epigenetic changes before and after growth or illness in the same individuals (to reduce sample size).
4. Survey the prevalence of rare and infectious diseases in pediatric population (even leftover blood samples unlinked to EHR may be used for this purpose).
5. Identify populational genetic polymorphisms that are not compounded by aging (even leftover blood samples unlinked to EHR may be used for this purpose).
1. Z. Chen. Residual blood samples: medical waste or research treasure? New Horizons in Translational Medicine 1(1):6-7, 2013.