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Completely automated ambient temperature biobanking. Workflow for sample collection, transport
and storage of human blood samples for molecular RNA and DNA diagnostics.
Vasco Liberal, Angela Stassinopoulos, Scott Whitney, Steven Wilkinson, Winnie Huang, Rolf Muller and Judy Muller-Cohn
Biomatrica, Inc., 5627 Oberlin Drive, Suite 120, San Diego, CA 92121
Results
Abstract
Analysis of genomes and gene expression profiles are being
increasingly used for diagnosis and monitoring of disease, and require
reliable nucleic acid preservation during sample collection and shipment.
Transcription profiles can change rapidly after sample collection,
potentially affecting interpretation of gene expression and ultimately
dictating inadequate treatment. Biomatrica’s RNAgard® Blood Tube is a
device for whole blood collection, which stabilizes both the RNA and
DNA in blood cells and allows for ambient temperature blood sample
handling and storage. Coupled with robust purification methods for both
nucleic acids, it provides a complete solution for blood collection and
storage, yielding both high quantity and quality of RNA and DNA. RNA
isolation can be performed with multiple automated platforms, allowing
ease and flexibility in the workflow and excellent results for low to high
throughput sample processing. Moreover, the RNAgard Blood Tube
allows for the isolation of both DNA and RNA from the same patient
sample, simplifying logistics, especially when large sample sets are
required. Isolation of RNA and DNA from the same sample is ideal, since
the use of separate samples could introduce operation errors and
variation of results. We have therefore developed an automated
workflow for sequential RNA and DNA purification, using the MagNA
Pure System (Roche). Biomatrica’s ambient temperature nucleic acid
preservation technology, coupled with automated purification, can
provide accurate gene expression profiles, proving highly valuable for
improved biomedical research and potential patient treatment.
Collection
Transport
Process
Archive
RNA Isolation using the automated MagNA Pure
System
Figure 1: High yields of pure RNA from blood specimens in
RNAgard Blood Tubes using the MagNA Pure Compact System.
Blood from two human donors was collected in RNAgard Blood Tubes
and stored at ambient temperature for 14 days. RNA was isolated from
triplicate blood specimens per donor on the MagNA Pure System, and
was analyzed for RNA yield (A) and RNA purity (A260/A280) (B) .
DNA Isolation using the automated MagNA Pure
System
Figure 4: High integrity and yield of genomic DNA isolated by the
MagNA Pure System from blood stored in RNAgard Blood Tubes.
Triplicate blood samples per donor from two donors was analyzed at
Day 0 and after 14 days of ambient temperature storage. The DNAcontaining supernatant recovered after RNA pelleting (see methods)
was processed for DNA isolation. DNA integrity was assessed by
agarose gel electrophoresis (A) and yield was measured by UV
specrophotometry (B).
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Materials and Methods
Blood collection and storage: Human blood from two healthy donors
was collected in RNAgard Blood Tubes. Nucleic acid isolation was
performed immediately upon blood arrival in the laboratory (Day 0). The
remaining blood tubes were stored at ambient room temperature for 14
days before processing.
Automated nucleic acid isolation: RNA isolation: Three RNAgard Blood
Tubes were processed for nucleic acid isolation per donor at each time
point. RNA was pelleted in blood samples by the addition of BioMaxi™
Precipitation Buffer followed by centrifugation according to the standard
procedure for the RNAgard Blood System. The DNA-containing
supernatant was saved for separate DNA purification. The RNAcontaining pellet was resuspended in 200 µL of lysis buffer from the
MagNA Pure Compact RNA Isolation Kit, and RNA was purified using the
MagNA Pure Compact System (Roche Applied Sciences). DNA
isolation: The DNA-containing supernatant was processed for DNA
isolation on the MagNA Pure Compact instrument with the MagNA Pure
Compact Nucleic Acid Isolation Kit I.
RNA analysis: RNA integrity was assessed by agarose gel
electrophoresis and Agilent 2100 Bioanalyzer. Total RNA yield and purity
were measured by UV spectrophotometry. Gene expression levels after
14 days of blood storage of c-Fos and IL-1β were normalized to 18s
rRNA and quantified by the ΔΔCt method relative to transcript levels at
Day 0. Genomic DNA contamination levels were quantified by qPCR
amplification of an RNaseP amplicon.
DNA analysis: DNA integrity was analyzed by agarose gel
electrophoresis and by long-range PCR amplification of a 7.5 kbp region
from the β-globin gene followed by electrophoresis. DNA yield and purity
were quantified by UV spectrophotometry.
Figure 2: High integrity of RNA isolated from RNAgard Blood Tubes
after 14 days of ambient temperature storage. The integrity of RNA
recovered on the MagNA Pure System from triplicate blood specimens
per donor was assessed using an Agilent 2100 Bioanalyzer. Average
RIN values (A), gel-like images (B) and electropherogram data (C) are
shown.
Figure 5: Spectrophotometric analysis of genomic DNA purity and
assessment of DNA integrity by long-range PCR. The purity of DNA
isolated from triplicate RNAgard Blood Tube specimens per donor was
quantified by UV spectrophotometry (A). DNA integrity from the 14 day
old RNAgard Blood samples was assessed by long-range PCR
amplification of a 7.5 kbp region of the β-globin gene and compared to
amplification from fresh blood DNA.
Summary
Ambient temperature blood storage
automated nucleic acid isolation:
coupled
with
• RNA and DNA stabilized in human blood for 14 days at
ambient temperature in RNAgard Blood Tubes
Figure 3: Transcript stability and low genomic DNA content in RNA
samples isolated from RNAgard Blood Tubes. Transcript stability after
14 days of ambient temperature blood storage was verified by
comparison to c-Fos and IL-1β levels at the time of blood collection (time
0). Transcript levels were quantified by RT-qPCR using 18s rRNA as the
reference gene (A). Genomic DNA content in purified RNA samples was
quantified by qPCR amplification of an RNaseP target as a function of
input nucleic acid.
• Automated RNA and DNA isolation from RNAgard
Blood Tubes (Biomatrica) using the MagNA Pure
System (Roche Applied Sciences)
o RNA
• Stable transcription profile
• Yield > 3 µg/ tube
• Superior quality and purity
o DNA
• Excellent purity
• Highly intact
• Yield > 10 µg/ mL blood