Isolation of microarray-grade total RNA, microRNA, and DNA from a single PAXgene blood RNA tube

Abstract

We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated microRNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis.

Figure 1

Overview scheme for the TRI-X workflow. TRI-X is a combination of three methods, starting with 2.5-ml whole blood collected in a single PAXgene Blood RNA tube. Genomic DNA is isolated from a 1-ml aliquot with the QIAamp Blood DNA protocol. The remaining sample is extracted with the PAXgene Blood RNA MDx procedure to isolate mRNAs and large rRNA fragments. Small RNA species including miRNA are isolated from the flow through of the PAXgene Blood RNA MDx protocol binding step via a modified RNeasy procedure.

Figure 2

Sample mismatch analysis showing IBS scores of chip to chip comparisons of 250K SNP array. Heat map showing the result of a sample mismatch analysis comparing all SNP arrays against each other. The color of each square indicates the IBS score between SNP samples ranging from 0.9 to 2. The gray boxes indicate the average IBS score and SD between each group. Cnt, control DNA.

Figure 3

Electropherograms from Agilent Bioanalyzer. The y axis represents fluorescence units [FU], and the x axis represents the runtime (s). A: Typical picture of a total RNA preparation with the PAXgene Blood RNA MDx protocol. The bands of the 18S and 28S rRNA fragments are clearly visible, and the RNA integrity number score is 8.7. Only very limited amounts of small RNA species are visible. B: Typical picture of a small RNA preparation isolated from the flow through from the PAXgene Blood RNA MDx binding step. Small RNAs including miRNA are clearly enriched.