RNAdvance Blood Performance and Data

Total RNA Isolation and Purification from Blood

The RNAdvance Blood system is a ribonucleic acid (RNA) isolation process built on SPRI paramagnetic bead-based technology. It enables purification of high-quality RNA from blood collected using PAXgene tubes. Total RNA extracted from PAXgene-preserved blood using the RNAdvance Blood kit is free of detectable genomic DNA (gDNA).

  • Compatible with downstream gene expression analysis techniques:
    • NGS
    • qRT-PCR
    • Microarray
  • Produces high-quality RNA
  • Efficient removal of gDNA and other contaminants

 

RNA Integrity Consistent Among Samples

RNAdvance Blood isolates RNA with high RIN scores.

Samples were from three different tubes taken from the same donor. RNA isolated from blood preserved in PAXgene tubes were run on an Agilent 6000 RNA Nano Assay using a Bioanalyzer (Agilent Technologies). The RIN scores averaged 9.4 with a σ2 of 0.4 for the 9 samples prepped.

RNAdvance RNA Integrity Consistency - Tube 2a

Sample RIN
Tube 1a 8.6
Tube 2a 9.5
Tube 3a 9.4
Tube 1b 9.4
Tube 2b 9.9
Tube 3b 9.3
Tube 1c 9.8
Tube 2c 9.9
Tube 3c 8.7

RNAdvance RNA Integrity Consistency - Tube 1b

RNAdvance RNA Integrity Consistency - Tube 1c

 

 

RNAdvance Blood Isolates RNA at a Higher Yield Than Other Commercially Available Kits While Maintaining RNA Purity

RNA was extracted from blood preserved in PAXgene tubes according to manufacturer instructions.

 
RNAdvance Blood Yield RNAdvance Blood Purity

(Left) Samples were quantified using the NanoDrop (Thermo Fisher Scientific). RNAdvance blood gave the highest RNA yield compared to other commercially available kits.

(Right) Samples were assessed for purity using the NanoDrop (Thermo Fisher Scientific). All kits isolated RNA with purity ratios suitable for downstream applications.

 

RNAdvance Eliminates DNA and PCR Inhibitors for Use in Downstream Applications

RNAdvance Amplification Plot of RNA RNAdvance Amplification Plot of 10ng of RNA

(Left) The amplification plot of RNA extracted using the RNAdvance blood kit. The purple and black lines represent six replicates that used 10 ng and 1 ng of extracted RNA, respectively, as inputs for qRT-PCR; the gray lines represent negative controls where no reverse transcriptase were added. The ΔRN is the normalized reporter value. The difference in the purple and black lines indicates that there are no PCR inhibitors in the final RNA samples, and the difference between the gray lines and the purple and black lines indicates the removal of DNA in the final RNA samples.

(Right) The amplification plot of 10 ng of RNA extracted using RNAdvance blood and two other suppliers. The RNAdvance blood has lower Ct values than the two other suppliers. PCR amplifiability was assessed via qRT-PCR using a primer set (forward primer 5´-ggacttcgagcaagagatgg-3´ and reverse primer 5´-agcactgtgttggcgtacag-3´) designed to span Exon 4 and 5 of the beta (β)-actin gene (ActB) to produce 327 base pair amplicons.

 

Visual Workflow

RNAdvance Workflow

  1. Lyse PAXgene blood in Lysis Buffer and proteinase K
  2. Bind RNA to magnetic beads
  3. Separate magnetic beads from contaminants
  4. Wash magnetic beads with Wash Buffer and 70% ethanol to remove contaminants
  5. Treat samples with DNase I
  6. Rebind RNA to magnetic beads with Bind 2 Buffer
  7. Wash magnetic beads with 70% ethanol to remove contaminants
  8. Elute RNA from magnetic beads
  9. Transfer to new plate

 

Automation vs. Manual Timing

Estimated hands-on time and total time in hours required to perform 8, 24, 48 and 96 RNAdvance Blood RNA extractions. Extractions can be performed either manually or automated on a Biomek  Workstation. The table represents automation times performed on a Biomek i7 Hybrid. The difference in time between manual and automation is indicated. NR=Not Recommended.

 

RNAdvance Blood Manual Automated
Batch Size 8 Hands-on Time 1.00 0.25
Total Time 2.50 2.50
24 Hands-on Time 1.50 0.25
Total Time 3.00 2.50
48 Hands-on Time NR 0.25
Total Time NR 2.00
96 Hands-on Time NR 0.25
Total Time NR 2.50

 

Products for Extracting RNA from Blood

PART # NAME PREPS
A35603 RNAdvance Blood Kit  50
A35605 RNAdvance Blood Kit  96
A35604 RNAdvance Blood Kit  384

 

 

Not intended or validated for use in the diagnosis of disease or other conditions.
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