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Comparison of blood RNA isolation methods from samples stabilized in Tempus tubes and stored at a large human biobank.

Author(s): Aarem J, Brunborg G, Aas KK, Harbak K, Taipale MM, Magnus P, Knudsen GP, Duale N

Publication: BMC Res Notes, 2016, Vol. 9, Page 430

PubMed ID: 27587079 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared yield, purity, and integrity among RNA extracted from adult blood and umbilical cord blood using five different kits. Blood was collected from four adult volunteers into 59 Tempus tubes and from three umbilical cords into 18 Tempus tubes and stored for 2 h at room temperature, followed by overnight storage at -20˚C, and then transferred to -80˚C. Specimens were thawed and total RNA was extracted using the MagMAX semi-automated, MagMAX manual, Norgen, Tempus Spin, and Tempus 6-port kits. RNA yield was determined by spectrophotometry and integrity was determined using a bioanalyzer. Levels of six mRNAs and 13 miRNAs were determined by real-time PCR in specimens extracted using MagMAX or Norgen kits and miRNA levels were normalized to RNU6, RNU43, and RNU1.

   Summary of Findings:

   The RNA yield was significantly higher from the umbilical cord blood than from the blood of adults (P<0.05). The RNA yield from adult blood was comparable between isolation kits, but using either of the MagMAX methods resulted in higher RNA yields from umbilical cord blood than using the Norgen, Tempus Spin, or Tempus 6-port methods (P<0.05, all). As determined by an OD 260/230 ratio of less than 1.8, RNA isolated from adult blood using the MagMAX manual method or Norgen kit was contaminated (OD260/230 of 1.19 ± 0.12 and 1.76 ± 0.07, respectively) and significantly lower than the OD 260/230 obtained from umbilical cord blood or from adult blood using the other extraction kits. Nevertheless, the purity (OD 260/280) was acceptable for all specimens and unaffected by extraction method. RIN was not significantly affected by extraction method but the highest RIN was obtained from adult specimens using the Tempus Spin kit and from cord blood using the Tempus 6-port. CT values for the six mRNAs were similar, regardless of extraction kit used, with overall CVs between methods of 1.5-2.8 for adult specimens and 1.8-3.9 for umbilical cord blood. Although raw CT values for the 13 miRNAs differed slightly between the methods, they were comparable using the Tempus kits (which are not recommended for isolation of RNA less than 200 nt) to other methods and the between-method CVs weren’t significantly higher than the variability within a protocol. Nevertheless, Tempus kits were excluded from further analysis of miRNA.  CVs of miRNA levels were generally lower in RNA from umbilical cord blood than adult blood and were lower when extracted using the Norgen kit than the MagMax kits. Normalized miRNA levels in adult blood and umbilical cord differed slightly among the extraction methods, but were very strongly correlated among the three methods (r=0.91-0.98).

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant
   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	MagMAX semi-automated MagMAX manual Norgen Tempus Spin Tempus 6-port

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