Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples.
One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.
Chai
Viengthong
V
Lorus Therapeutics Inc, Toronto, Ontario, Canada.
Vassilakos
Aikaterini
A
Lee
Yoon
Y
Wright
Jim A
JA
Young
Aiping H
AH
D000199:Actins / Q000096:biosynthesis; D001800:Blood Specimen Collection / Q000379:methods*; D003851:Deoxyribonucleases; D004355:Drug Stability; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D009691:Nucleic Acid Denaturation; D012313:RNA / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012264:Ribonucleotide Reductases / Q000096:biosynthesis
D000199:Actins; D012313:RNA; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D003851:Deoxyribonucleases