blood RNA,RNA extraction,biospecimen,DNA extraction,cell viability ,preservative,random mutagenesis rocky
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Total blood DNA and RNA 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.AquaPlasmid, AquaGenomic, AquaRNA, AquaStool, AquaPreserve, AquaBluer 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



 AquaPreserve *

RNA yield 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
  $0.45/standard vacutainer
DNA yield 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


Centrifugation steps per extraction 11 (RNA) + 4 (DNA) = 15 3
Recover total DNA, RNA and proteins



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)

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