Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples.

Author(s): Chai V, Vassilakos A, Lee Y, Wright JA, Young AH

Publication: J Clin Lab Anal, 2005, Vol. 19, Page 182-8

PubMed ID: 16170815 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of blood collection container, storage at room temperature, RNA extraction method, and DNAse treatments on RNA quantity and quality. Blood collected in K2 EDTA vacutainers was stored for 3 h at room temperature prior to erythrocyte lysis, storage of leukocytes at -80 degrees C for 6 days, and extraction of RNA from leukocytes using Trizol. The remaining samples were collected into PAXgene tubes and stored for 24 h or 5 days at room temperature prior to RNA extraction using the PAXxgene blood RNA kit.

   Summary of Findings:

   The RNA yield was non-significantly higher when blood was collected in PAXgene tubes, stored for 24 h, and RNA was extracted using PAXgene than when blood was collected and extracted by the standard method or stored for 5 days at room temperature prior to PAXgene extraction. When blood was collected and RNA was extracted using standard methods, the RNA was degraded to lengths between 0.2 and 2 kb and the 18S and 28S ribosomal RNA bands were completely absent. In contrast, RNA was intact in specimens collected and extracted using PAXgene. When the specimens were stored at room temperature for 5 days rather than 24 h, the amount of intact 18S and 28S ribosomal RNA was lower. There was no DNA contamination of the specimens when RNA was extracted with Trizol followed by two rounds of DNA digestion with DNAse-free, but RNA extracted using the PAXgene kit with on column DNAse treatment was contaminated with DNA. Subsequent treatment of PAXgene-isolated DNA with the DNAse-free kit removed the contaminating DNA without affecting RT-PCR amplification of R1, R2, or beta-actin. There was greater amplification of R1, R2, and beta actin in specimens isolated by PAXgene than by the standard method. The amplification of beta actin was particularly reduced in specimens isolated by the standard method rather than by PAXgene, so RNA levels of R1 and R2 normalized to beta-actin appeared higher in RNA isolated by the standard method rather than by PAXgene. While a slight reduction in amplification of R1, R2 and beta actin was observed in specimens stored for 5 days rather than 24 h at room temperature prior to PAXgene isolation, this did not affect the normalized levels of R1 or R2.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • PAXgene

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Electrophoresis
   RNA	Spectrophotometry
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh) PAXgene
   Biospecimen Acquisition	Type of collection container/solution	K2 EDTA vacutainer PAXgene tube
   Storage	Time at room temperature	3 h 24 h 5 days
   Analyte Extraction and Purification	Analyte isolation method	Trizol PAXgene
   Analyte Extraction and Purification	Nucleic acid digestion	On column (PAXgene) DNAse-free (Ambion)
   RT-PCR Specific	Targeted nucleic acid	Ribonucleotide reductase subunit R1 Ribonucleotide reductase subunit R2 Beta actin

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