Stabilization of mRNA expression in whole blood samples.
BACKGROUND
Accurate quantification of mRNA in whole blood is made difficult by the simultaneous degradation of gene transcripts and unintended gene induction caused by sample handling or uncontrolled activation of coagulation. This study was designed to compare a new blood collection tube (PAXgene Blood RNA System) and a companion sample preparation reagent set with a traditional sample collection and preparation method for the purpose of gene expression analysis.


METHODS
We collected parallel blood samples from healthy donors into the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for 5 days at room temperature and for up to 90 days at 4 and 20 degrees C. Samples were analyzed by Northern blot analysis or reverse transcription-PCR (RT-PCR).


RESULTS
Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors.


CONCLUSIONS
Compared with whole blood collected in EDTA tubes and extracted by an organic method, the PAXgene Blood RNA System reduced RNA degradation and inhibited or eliminated gene induction in phlebotomy whole blood samples. Storage of whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.
Rainen
Lynne
L
PreAnalytiX (CH) c/o Becton Dickinson, Franklin Lakes, NJ 07417, USA. lynne_rainen@bd.com
Oelmueller
Uwe
U
Jurgensen
Stewart
S
Wyrich
Ralf
R
Ballas
Cynthia
C
Schram
Jim
J
Herdman
Chris
C
Bankaitis-Davis
Danute
D
Nicholls
Nancy
N
Trollinger
David
D
Tryon
Victor
V
D001800:Blood Specimen Collection / Q000295:instrumentation; D015152:Blotting, Northern; D004587:Electrophoresis, Agar Gel; D020869:Gene Expression Profiling; D006801:Humans; D012313:RNA / Q000032:analysis / Q000302:isolation & purification; D012333:RNA, Messenger / Q000097:blood*; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic; D012313:RNA