tempus blood rna tubes and rna isolation … · rna isolation chemistry applied biosystems has...

• Collect 3 mL of whole blood and stabilize RNA and transcriptprofiles for up to five days atroom temperature

• Isolate pristine-quality total RNA with very low genomic DNA levels, while maintaining pre-blood draw expression levels

• Obtain RNA suitable for real-timePCR, microarray analysis, or anyother procedure

• Simplified isolation protocol withvery little “hands-on” time

• Purify RNA from up to sixTempus Blood RNA CollectionTubes simultaneously in 45 minutes

IntroductionMeasuring gene expression in wholeblood is becoming an increasinglyimportant research tool. “Global”measurement of expression changes

in whole blood by microarray technologies permits the profiling of many thousands of mRNA transcripts simultaneously in a singlesample. Then, at the next phase of experimentation, 5' nuclease assays based on TaqMan® or SYBR®

Green 1 Dye reagent-based chemistryusing real-time PCR instrumentationcan be performed on a smaller numberof mRNA transcripts in a larger population group to understand theimportance of these expression

changes. Following these mRNA tran-scripts (also known as biomarkers),which have implications in diseasestates or treatment regimes, can be of

significant help in gaining an in-depthmolecular profile of a disease, orunderstanding the impact of various

treatments.

Isolation of high-quality RNA from

whole blood samples is difficult, andrecent work at Applied Biosystems(Tanner et al., Clin. Lab. Haem. 2002,24, 337–341) has shown that the task is complicated even furtherbecause under ambient conditions,expression profiles are unstable on atimescale of less than one hour. Thisinstability is the result of metabolic

activity that continues in standardblood collection tubes post-draw (see Figure 1). Up-regulation of ~50-fold has been measured for somegene targets after one hour at room

temperature. Because blood is oftendrawn at sites remote from the investi-gating team, there is often a time lag(typically hours but sometimes days)before the RNA isolation process can begin. During this time lag,

ex vivo changes in expression profiles

can occur.

Our results show that blood storage

under ambient conditions and lack ofoxygenation can significantly alter theexpression patterns of several impor-tant gene targets (see Tanner et al. andFigure 1). For accurate measurement of gene expression profiles, therefore,

blood must be drawn into a collectiontube that contains a stabilizing reagent.This permits immediate freezing and

stabilization of the profile for severaldays to allow for transportation delays.

The Tempus Blood Tube system isdesigned for direct isolation of 3 mL

Tempus™ Blood RNA Tubes and RNA Isolation ConsumablesA Complete System for Stabilizing and Isolating Total RNA from Whole Blood

for Gene Expression Analysis on the ABI PRISM™ 6100 Nucleic Acid PrepStation

Product Bulletin Sample Preparation Systems

www.appliedbiosystems.com

of patient blood into a plastic, evacuatedblood collection tube containing theApplied Biosystems RNA StabilizingReagent. The RNA, and therefore the expression or transcript profile, is stable in the collection tube for up to five days at room temperature(18–25°C), or longer at 4°C. The

stabilized blood may also be frozen

in the collection tube for prolongedstorage or transport.

A simple, 45-minute protocol on theABI PRISM 6100 PrepStation purifies

the RNA. The protocol requires asmall number of simple, hands-onsteps but generates high-quality nucle-ic acid (A260/280 > 1.9) at yields of 2–8 µg/mL of human whole blood.During isolation, an “in-protocol/on-

column” DNase treatment removes contaminating genomic DNA to levelsof < 0.002% by weight as measured by

real-time PCR. The 6100 PrepStationcan process up to six tubes simultane-

ously, producing total RNA that is free of RT/PCR inhibitors and otherenzymes. The RNA is also suitable forall downstream applications, includingreal-time PCR and microarray analysis.

Safe, High-Quality VACUETTE®

TubesThe Tempus Blood RNA Tube wasdeveloped collaboratively by AppliedBiosystems and Greiner Bio-OneGmbH, one of the world’s leading suppliers of blood collection products.Greiner Bio-One’s VACUETTE brandof blood collection tubes is wellknown for both quality and safety.

These blood collection tubes were the first to be manufactured from the virtually unbreakable polyethyleneterephthalate (PET) plastic. GreinerBio-One has also introduced a wide

variety of other innovations for the collection of blood and other sample types.

The Tempus Blood RNA Tube is

manufactured with a distinctive bluecap and black inner ring to distinguishit from other common venous blood-collection tubes. It contains 6 mL of RNA Stabilizing Reagent and is calibrated to draw 3 mL of wholeblood accurately. PET plastic tubes are

safer to use and transport than glasstubes, and they more easily withstandfreeze/thaw procedures.

Comprehensive details, includingsafety information on venous blood-collection techniques and a list of blood-collection accessories (butterflies, holders, needles, etc.) forVACUETTE blood collection prod-

ucts, are available at www.vacuette.com.You may also contact your local

distributor of VACUETTE productsfor additional information.


Figure 1. Gene expression stability in unstabilized whole blood (Tanner et al., Clin. Lab. Haem. April 2002, 24, 337–341)

IL-8c-mycc-fosG-CSFMCP-1MIP-1-alpha

IL-7LT-betaFASFasLp53CCR-3MCP-2

4 degrees 1

hr

4 degrees 4

hr

4 degrees O

/N

Ambient 1 hr

Ambient 4 hr

Ambient O/N

4 degrees 1

hr

4 degrees 4

hr

4 degrees O

/N

Ambient 1 hr

Ambient 4 hr

Ambient O/N

Genes Up-Regulated

Genes-Down Regulated

RNA Isolation ChemistryApplied Biosystems has developed a unique RNA extraction mechanismthat generates the highest quality totalRNA and requires only a small number of steps. It is the first novelpurification chemistry to be developedspecifically for RNA isolation in thelast 10 years. The reagent, which contains guanidine and detergent, stabilizes RNA by immediately lysingthe blood cells and inactivatingRNases through its chaotropic proper-ties. The detergent encapsulates thetotal RNA in a micelle structure andselectively precipitates it, leaving protein and genomic DNA in solution. The lysate is then filteredthrough a porous membrane that captures the RNA. Genomic DNA,protein, and other cell debris passthrough the membrane with very

low retention.

During purification, a DNase treatment can be performed on the

membrane to remove even the very low

levels of contaminating genomic DNAthat are expected. This results in levels

of < 0.002% by weight, or close to the

background level detectable by the 5'nuclease TaqMan-based assay. The onlyfurther manipulation required is a

dilution of the blood lysate with PBSbefore processing. This simple, stream-lined protocol includes very fewhands-on steps and requires no RBC

lysis, proteinase K treatment, heating,or prolonged incubation.

Applied Biosystems RNA chemistry

isolates a representative and proportionalselection of messenger, ribosomal, andsmall nuclear RNAs. It does not isolatetRNA, mammalian 5S ribosomalRNA, highly degraded RNA, or anyRNA species less than approximately200 nucleotides. This fact, along withvery low protein contamination of the

RNA, provides distinct advantagesover other extraction chemistries, inwhich tRNA and 5S ribosomal RNAoften comprise 15–20% of total RNA.

The RNA isolated by Applied

Biosystems RNA chemistry is highlystable, as it contains very little carry-over protein. Hence, there is almost

no RNase activity to contaminate theisolated nucleic acid. Nor are there any inhibitors of RT, PCR, or other

enzyme reactions. Taken together,these factors help ensure that the RNAyou obtain is pure, stable, and will

provide high-quality data for your real-time PCR or microarray applications.

Large-Volume RNA Isolation onthe ABI PRISM 6100 PrepStationPurification of total RNA from largevolumes of whole blood requires theuse of a specialized set of consumables

to handle the increased volumes of stabilized blood from the TempusBlood RNA Tube. The specialized

consumables consist of a Large-Volume Adaptor Plate for the vacuumcarriage of the 6100 PrepStation; aLarge-Volume RNA Prep Filter to cap-ture RNA from the stabilized whole blood; 20 mL and 5 mL reservoirs forloading the blood lysate onto the filterconsumable; DNase and wash reagentsto remove DNA, protein, and other

cellular contaminants; and a 2 mLTube Collection Plate for collecting


Figure 2. Comparison of gene expression stability from blood stabilized in Tempus Blood RNA Tubes and standardEDTA blood collection tubes.

Tempus

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EDTA1000.00

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the eluted RNA in a 2 mL microcen-trifuge tube. Alternatively, acentrifugation-based elution methodmay be used to provide RNA of

higher concentrations.

Applied Biosystems total RNA isolation reagents include Wash

Solution 1, Wash Solution 2,AbsoluteRNA Wash Solution (DNasetreatment), and Elution Solution.RNA preparations are expected toyield between 2 and 8 µg/mL ofhuman whole blood with high-qualitytotal RNA, having A260/280 ratios of

> 1.9. Typical yields and purity, there-fore, range between 6 and 24 µg oftotal RNA at A260/280 ratios of > 2.0(Figure 4).

Transcript StabilityWhen blood is drawn into a Tempustube, the blood cells are immediatelylysed, RNases are inactivated, andmost metabolic activity ceases.RNA is stabilized ensuring thatthe transcript profile measured at

blood draw accurately reflects thephysiological state of the patient. Forhuman samples, transcript profiles areexpected to be stable for up to five

days if stored at < 25°C (Figures 2

and 3). Storing the profiles at 4°Cimproves stability; they can be storedat this temperature for at least sevendays. Samples stored at from -20°C to-80°C should be stable for an extendedperiod of time. To ship tubes contain-

ing stabilized blood from remote sites, we recommend freezing the tube at

-80°C whenever possible, because

temperatures during transportation are not well regulated.

Figure 3. Agarose gel (1%) images of total RNA isolated after storage in Tempus and EDTA tubes at room temperature.

Legend: Lane M=2 µg RNA Millenium Markers. Lanes 0, 1, 3, 5, and 7=2 µg RNA from human whole bloodafter 0, 1, 3, 5, and 7 days storage at room temperature in Tempus (A) or EDTA (B) tubes.

B. EDTAA. Tempus

DaysM 0 1 3 5 7

Days0 1 3 5 7

Tempus Blood RNA Tubes and RNA Isolation Consumables—your complete solution for stabilizing and isolatingtotal RNA from whole blood for gene expression analysis.


Sample A260/280 Ratio Concentration ng/µL Yield µg of RNA per tube

Concentration 1 2.09 104 10.4




Figure 4. UV absorbance measurements of yield and purity of RNA from Tempus tubes.

iScience. To better understand the complex interaction of biological systems, life scientistsare developing revolutionary approaches to discovery that unite technology, informatics, and traditional laboratory research. In partnershipwith our customers, Applied Biosystems provides the innovative products, services, and knowledge resources that make this new,Integrated Science possible.

Worldwide Sales OfficesApplied Biosystems vast distribution and servicenetwork, composed of highly trained support andapplications personnel, reaches 150 countries onsix continents. For international office locations,please call the division headquarters or refer toour Web site at www.appliedbiosystems.com

Applera is committed to providing the world’sleading technology and information for life scientists. Applera Corporation consists of theApplied Biosystems and Celera Genomics businesses.

Headquarters850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: 650.638.5800Toll Free: 800.345.5224Fax: 650.638.5884

For Research Use Only. Not for use in diagnostic procedures.

Applied Biosystems is a registered trademark and AB (Design),ABI PRISM, iScience, iScience (Design), and Tempus are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.

TaqMan is a registered trademark of Roche MolecularSystems, Inc.

VACUETTE is a registered trademark of Greiner Bio-One GmbH.

SYBR is a registered trademark of Molecular Probes, Inc.

The PCR process and 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc and F. Hoffmann-La Roche Ltd.

©2004 Applied Biosystems. All rights reserved.

Printed in the USA, 04/04 P+s Publication 117PB15-01

Ordering InformationNote: The VACUETTE® Safety Blood Collection Sets listed below are not supplied by Applied Biosystems and must be ordered directly from Greiner Bio-One GmbH or your Greiner Bio-One distributor. For subsidiary/distributor information, please refer to www.gbo.com/en/

VACUETTE® Safety Blood Collection Products

Description Tube length Gauge Quantity P/N

Safety Blood Collection Sets* 19 cm 21 G 24/240 450085√




Safety Blood Collection Sets** 30 cm 23 G 50/1,000 450096

Tube Holder for P/N 450096*** ---- ---- 10/1,200 450201*Safety Blood Collection Sets with Luer Adapter and sterile tube holder (24 pcs. in the inner pack and

240 pcs. in total)

**Safety Blood Collection Set just with Luer Adapter (50 pcs. in the inner pack and 1,000 pcs. in total)

***Tube Holder (10 pcs. in the inner pack and 1,200 pcs. in total)

√Not available in the US

Applied Biosystems Consumables Quantity P/N

Tempus™ Blood RNA Tubes 50/box 4342792

Large-Volume Upgrade Starter Kit Each 4344440Includes: Large-Volume Adaptor Plate, 2 mL Tube Collection Plate, 6 Large-Volume RNA Prep Filters, 6 Reservoirs (20 mL each), 18 Reservoirs (5 mL each); sufficient for 6 large-volume purifications

Large-Volume RNA Prep Filters 50/box 4344439

20 mL Reservoir 50/box 4344435

5 mL Reservoir 150/box 4344437

Large-Volume Adaptor Plate Kit Each 4344438Includes: 20 disposable Adaptor Plates, 120 Microelution vials, 10 Slip-Fit Cap Plugs

2 mL Tube Collection Plate Each 4344436(For the Collection position on the ABI PRISM™ 6100 PrepStation)

2 mL Microcentrifuge Tubes 500/box 4305936

Splash Guards 20/box 4311758

Reagents

AbsoluteRNA Wash Solution 10 mL 4305545DNase Reagent, sufficient for removal of contaminating genomic DNA from a minimum of 30 Tempus tubes to a level of < 0.002% by weight in 15 minutes

RNA Wash Solution 1 1 L 4305891

RNA Wash Solution 2 1 L 4305890

RNA Elution Solution 1 L 4305893

High-Capacity cDNA Archive Kit Each 4322171Converts up to 10 µg of total RNA to single-stranded cDNA in one 100 µL reaction, using a random priming scheme. Each kit contains sufficient reagents for 200 reactions (100 µL each)

Instrument

ABI PRISM™ 6100 Nucleic Acid PrepStation Each 6100-01

tempus blood rna tubes and rna isolation … · rna isolation chemistry applied biosystems has...

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