Recovery of microarray-quality RNA from frozen EDTA blood samples.
BACKGROUND
The availability of blood collection systems with RNA stabilizing additives (e.g. PAXgene) has opened the field for gene expression profiling in large multi-center clinical trials. Here, we investigated whether the PAXgene system also offers a method for extraction of RNA from frozen EDTA blood samples, which do not yield RNA of high enough quality for RNA expression profiling, when extracted with standard protocols.


METHODS
Whole blood was obtained from six healthy volunteers in conventional EDTA tubes and frozen. The thawed EDTA blood was transferred into PAXgene tubes, and the RNA was extracted using the PAXgene RNA extraction kit. Microarray analysis was performed to asses the effect of RNA quality on gene expression profiles.


RESULTS
The RNA yield of the transferred samples was 1.76+/-0.88 microg/ml. This yield was clearly lower than the yield from a PAXgene reference group (2.84+/-0.62 microg/ml), but considerably higher than the yield resulting from a standard protocol usually applied to fresh EDTA blood samples (0.07+/-0.06 microg/ml). The RNA integrity number (RIN) of the transferred samples was 6.1+/-0.8 as compared to 9.8+/-0.1 for the PAXgene reference. Microarray analysis of the extracted RNA suggested that samples with RIN values above 5 produce data that fulfill the quality criteria defined by the manufacturer.


CONCLUSIONS
The transfer of thawed EDTA blood into PAXgene blood collection tubes offers a method to recover sufficient RNA of acceptable quality for microarray experiments.
Beekman
Johanna M
JM
Clinical Pharmacology, Bayer Schering Pharma AG, Berlin, Germany. johanna.beekman@bayerhealthcare.com
Reischl
Joachim
J
Henderson
David
D
Bauer
David
D
Ternes
Rainer
R
Peña
Carol
C
Lathia
Chetan
C
Heubach
Jürgen F
JF
D000328:Adult; D001793:Blood Preservation / Q000379:methods*; D001800:Blood Specimen Collection / Q000379:methods; D004492:Edetic Acid / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D008297:Male; D008875:Middle Aged; D008957:Models, Genetic; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011786:Quality Control; D012313:RNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods
D012313:RNA; D004492:Edetic Acid