SeraCare Technical Bulletin: High Quality RNA Extraction
Q: What are the best methods for high throughput and low
throughput extraction of RNA from PAXgene tubes?
A: SeraCare’s scientists regularly perform
RNA extractions in large and small sample numbers
and have found the QIAsymphony PAXgene
Blood RNA Kit with the QIAsymphony instrument
meets their needs for high throughput RNA extraction.
The QIAcube is SeraCare’s method of
choice when total RNA yield is critical or for low to
medium throughput requirements. SeraCare scientists
use both methods frequently and obtain
RNA of excellent quality and integrity.
RNA transcript levels can change dramatically,
often within minutes of blood collection, due to
gene induction, cell death, or RNA degradation.
PAXgene Blood RNA tubes from PreAnalytiX®
stabilize intracellular RNA at the point of blood
collection. According to the manufacturer, blood
collected in PAXgene tubes is stable for up to four
years when frozen at -20ºC and delivers consistent
yields of >3 μg RNA per tube with low levels of
contaminants.
High Throughput Extraction Using
the QIAsymphony
The Qiagen QIAsymphony SP is a biorobot used for
automated extraction of RNA from PAXgene tubes.
It is a highly efficient method, with bar code scanning
and full sample tracking throughout the entire
procedure. The system integrates well with
SeraCare’s sample storage system.
Filled PAXgene tubes are thawed (if necessary) and
centrifuged until the RNA-containing precipitate
forms a pellet. After the supernatant is decanted,
the PAXgene tube is loaded directly onto the
QIAsymphony instrument. Since there are no sample
transfer steps and the instrument scans sample
bar codes directly from the tubes, sample identity
is securely maintained throughout extraction.
To demonstrate performance consistency, samples
from four different donors were extracted in triplicate
during two extraction runs on the QIAsymphony.
Results indicate consistency in RNA yield for
samples extracted on the same run (Figure 1), with
a coefficient of variation (CV) of 7.2%. Between-run
CV was calculated at 13.1%, suggesting overall
consistency in RNA yields.
Low to Medium Throughput Extraction
Using the QIAcube
The Qiagen QIAcube is a biorobot used for semiautomated
extraction of RNA from PAXgene
tubes.The QIAcube uses the same kit (PAXgene Blood RNA Kit IVD) utilized for fully manual
extractions. Purification is based on spin column
technology, as opposed to the magnetic beads used
in QIAsymphony extraction.
For the QIAcube, filled PAXgene tubes are thawed
(if necessary) and centrifuged until the RNA-containing
precipitate forms a pellet. After the supernatant
is decanted, the pellet is resuspended in
water as a wash step, re-centrifuged, and then suspended
in buffer. This solution is transferred to a
processing tube and loaded onto the QIAcube. The
variation in RNA yield from four different donors extracted
on the QIAcube is consistent within run, with
a CV of 8.22% (Figure 2). The spin column technology
used in the QIAcube results in lower RNA loss
and maximizes yield compared to the QIAsymphony
(Figure 3).
Purity and Integrity of RNA from Both
Extraction Methods
The integrity of the RNA extracted using both the
QIAsymphony and QIAcube was analyzed using the
Agilent 2100 Bioanalyzer. A RIN number is an estimate
of RNA integrity and is determined by Agilent
software through analysis of the electrophoretic
trace, including comparison of the ratio of 18S and
28S ribosomal RNA bands, detection of RNA degradation
products, and other factors. RIN values of 7.0
or greater are considered excellent integrity. For the
24 samples (four donors) extracted on the QIAsymphony,
the average RIN was 7.27 (7.60% CV). Electropherograms
and the gel for Donor A demonstrate
high RNA integrity (Figure 4).
The RIN values for the samples extracted using the
QIAcube were very similar and very consistent with
a CV of 4.22%. RNA purified by both the QIAcube and
QIAsymphony had clear 18S and 28S ribosomal
RNA bands and was generally free of degradation
products. UV spectrophotometer analysis also indicated
that the RNA from both methods was free of
contaminants; the average A260/280 was 2.07
for QIAsymphony samples and 2.06 for QIAcube
samples.
To demonstrate that the extracted RNA is suitable
for further experimentation, TaqMan real time
reverse transcription PCR was performed on RNA
samples extracted with the QIAsymphony, using
primers and probes targeting a non-coding
sequence. All samples were detected with similar
Cycle threshold (Ct) values (Figure 5 Top). The amplification
curves from triplicate extractions of one
donor analyzed in duplicate by real time PCR indicate
the extracted RNA has a purity level acceptable
for PCR-based downstream applications (Figure 5
Bottom).
Analysis of Long Term Storage
Frequently, blood samples may be stored frozen for
an extended period of time before processing. To
examine the effects of long term storage in PAXgene
tubes on the purification of RNA with the
QIAsymphony, RNA yield from historical samples was analyzed. SeraCare obtained triplicate PAXgene
tubes from each of three donors; the tubes had been
collected in Germany in December, 2007, and stored
at -20ºC by Qiagen for approximately 3 years. They were extracted by SeraCare from these tubes in
November, 2010, using the QIAsymphony SP
instrument (Figure 6).
The average RIN value of these samples was 6.23
(10.91% CV). Although the average yield and
average RIN numbers are slightly lower than the
averages from newly collected PAXgene tubes, the
yields are still consistent with the PAXgene manufacturer’s claims of >3 μg RNA per tube and
the RNA integrity is acceptable for most downstream
applications.
Conclusion
As a regular part of services at SeraCare Life
Sciences, scientists routinely perform RNA extractions
from PAXgene tubes using both the
QIAsymphony and the QIAcube. Each method has
different advantages and disadvantages. Both
methods deliver RNA intact, free of contaminants,
and suitable for a variety of downstream applications.
For low to medium throughput extractions,
the QIAcube method allows semi-automated extraction
of RNA with high yield, purity, and integrity.
For high throughput applications, the QIAsymphony
is fully automated and readily scalable for sample
numbers reaching into the hundreds or thousands.
While some yield may be sacrificed by the more
automated method, the samples are still high
quality and show consistent results in RT-PCR,
demonstrating suitability for a variety of downstream
applications. Additionally, high throughput
extraction of RNA from blood stored for long
periods has been tested on this method, demonstrating
that RNA from whole blood samples stored
at SeraCare’s biorepository can be extracted and
further tested even after years of storage.
Related SeraCare Products and Services
- RNA extraction from PAXgene
Blood RNA tubes, whole blood,
and tissues
- DNA extraction from blood, buffy
coat, cultured cells, saliva, and
other biological specimens
- DNA and RNA normalization,
aliquoting, and plating into
96-well plates
- Biorepository and sample
management services