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Evaluation of RNA purification methods by using different blood stabilization tubes: identification of key features for epidemiological studies.

Author(s): Carrillo-Ávila JA, de la Puente R, Catalina P, Rejón JD, Espín-Vallejo L, Valdivieso V, Aguilar-Quesada R

Publication: BMC Res Notes, 2020, Vol. 13, Page 77

PubMed ID: 32070402 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the RNA yield, purity, and integrity as well as amplification of miRNA and mRNA in matched blood specimens collected in PAXgene tubes and extracted using the PAXgene Kit to those collected in Tempus tubes and extracted using the MagMax or Tempus kits. Blood was collected from 25 healthy donors into four PAXgene and eight Tempus tubes. In order to eliminate effects of technician, duplicate specimens were processed by a different technician such that each technician processed one PAXgene and two Tempus specimens from each of the 25 donors. RNA was extracted from PAXgene tubes using the PaxGene Blood miRNA Kit in conjunction with a Qiacube and from Tempus specimens using MagMax for Stabilized Blood Tubes RNA Isolation Kit using a DynaMag-2 stand and with the Tempus Spin RNA Isolation Kit without DNAse. RNA yield and purity were evaluated spectrophotometrically and RNA integrity was measured using the Agilent RNA 6000 Nano Kit on a bioanalyzer. RNA yield from 42 specimens (7 donors) was also quantified using the Quant-iT RiboGreen RNA Assay Kit. RNA from seven donors was reverse-transcribed using the miScript II RT Kit and miR16, miR26, and miR30 were quantified with the miScript SYBR Green PCR Kit. Levels of 18S rRNA, ACTB, and GAPDH were quantified by real-time PCR.

   Summary of Findings:

   The highest yield was obtained when blood was collected in Tempus tubes and extracted using the Tempus Spin RNA Isolation Kit (13.5 µg versus 9.76 for MagMax and 8.64 for PAXgene, P<0.05). The RNA yield differed between technicians using either extraction method when blood was collected in Tempus tubes but was comparable between technicians when collected and extracted using PAXgene. Although A260/A280 ratios were slightly better when blood was collected in PAXgene tubes and extracted with the PAXgene Kit or collected in Tempus tubes and extracted using the Tempus Kit compared to when collected in Tempus tubes and extracted using the MagMax Kit, the differences were not significant and there were no differences attributed to technician. Similarly, purity as evaluated by A260/A230 ratios was highest when RNA was extracted from blood in in Tempus tubes using the Tempus Kit and lowest when extracted from blood in Tempus tubes using the MagMax Kit and when extracted from blood in Tempus tubes using the MagMax Kit the ratios differed between technicians. RINS were highest when blood was collected in PAXgene tubes and extracted with the PAXgene Kit or collected in Tempus tubes and extracted using the Tempus Kit, but the differences were not significant and there were no differences between technicians. The authors noted some specimens from PAXgene tubes extracted with the PAXgene Kit had high concentrations of large fragments which could be eliminated by treatment with DNAse. The reproducibility in yield, purity, and RIN were highest when RNA was extracted from blood in Tempus tubes using the Tempus Kit, but the reproducibility in yield using this method depended on the technician. Interestingly, Cq values for the 18S rRNA gene and ACTB, and to a lesser extent GAPDH, were comparable between kits when extracted from blood in Tempus tubes, but the Cqs were much lower than for those in PAXgene tubes. In contrast, the lowest Cq was observed for miR-30 when blood was collected in PAXgene tubes and extracted using the PAXgene Kit and differences in miR-26 and miR-16 were less clear.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	Tempus tube PAXgene tube
   Real-time qRT-PCR Specific	Targeted nucleic acid	18S rRNA ACTB GAPDH miR-30 miR-16 miR-26
   Analyte Extraction and Purification	Nucleic acid digestion	DNAse treated Untreated
   Biospecimen Preservation	Type of fixation/preservation	PAXgene Tempus
   Analyte Extraction and Purification	Analyte isolation method	PAXgene kit Tempus kit MagMax kit

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