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Characteristics of RNA Stabilizer RNApro for Peripheral Blood Collection.

Author(s): Gambarino S, Galliano I, Clemente A, Calvi C, Montanari P, Pau A, Dini M, Bergallo M

Publication: Diagnostics (Basel), 2024, Vol. 14, Page

PubMed ID: 38786269 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated RNA stability by comparing RNA yield, integrity and GAPDH cycle threshold (CT) values of RNA extracted from case-matched blood stored in tubes containing RNApro stabilization solution at -80°C for 48 h versus up to 6 years; among RNA extracted from case-matched blood stored in tubes with RNApro solution at -80°C, -20°C, 4°C or room temperature for 1 week or 1 month; and between RNA extracted from blood stored in PAXgene tubes versus RNApro containing tubes. Additionally, the efficacy of RNApro was compared after storage of the stock solution at -80°C, -20°C, 4°C or room temperature for 2 months. Blood was collected from 40 patients (diagnosis not specified) into K2EDTA Vacutainer tubes containing 800 µL RNApro stabilization solution, and PAXgene Blood RNA tubes. For most experiments, RNApro stock solution was stored at -80°C until use; but, the stock solution was stored at -80°C, -20°C, 4°C, or room temperature for 2 months before use to assess its stability. To investigate RNA stability, RNA extracted from case-matched blood specimens stored in RNApro solution at -80°C for 48 h or 1-6 years (collected in 2017 to 2022) were compared, and case-matched blood specimens in RNApro solution that were stored at -80°C, -20°C, 4°C, or room temperature for 1 week or 1 month were compared. Ten specimens were used to compare blood stored for 24 h at -80°C in PAXgene RNA tubes with those stored in tubes containing RNApro solution. RNA was extracted from blood using a modification of the Maxwell RNA Blood Kit (no lysis step for RNApro specimens). RNA quantity and purity were evaluated by spectrophotometer and the remaining RNA was stored at -80°C until real-time RT-PCR amplification of GAPDH. DNA contamination was evaluated by direct amplification of GAPDH without reverse transcription. Levels of miR were quantified by real-time PCR using the GeneAmp RNA PCR Kit.

   Summary of Findings:

   RNA yields, purity (OD 260/280), and cycle threshold (CT) values for GAPDH were all comparable between case-matched blood specimens stored in RNApro stabilization solution at -80°C for 48 h before extraction and matched specimens stored at -80°C from collection (2017-2022) until analysis (2023) up to 6 years later. RNA yield was significantly lower when blood was stored with RNApro solution at room temperature for 1 week or 1 month than case-matched blood stored with RNApro solution for the same duration at -80°C (P=0.0036 and P=0.0067, respectively ), -20°C (P=0.0002 and P=0.0043, respectively), or 4°C (P=0.0005 and P=0.0103, respectively); RNA yields were comparable among RNApro blood specimens stored at -80°C, -20°C, or 4°C. There was a correspondingly higher CT value for GAPDH after 1 week or 1 month of room temperature storage with RNApro solution compared to values from RNApro specimens stored at -80°C, -20°C, or 4°C (P<0.0001, all), but the CT values were also higher after RNApro blood was stored for 1 month (but not 1 week) at 4°C than RNApro blood stored at -80°C or -20°C (P≤0.0001, all). When the RNApro stock solution was stored at room temperature rather than -80°C, -20°C or 4°C, RNA yields decreased in blood stored at -80°C for 48 h prior to analysis (P=0.0312, P=0.0107, and P=0.0254, respectively) but GAPDH CT values were unaffected. RNA from blood stored with RNApro solution, as opposed to PAXgene tubes, had a higher mean yield (25.7 ng/µL versus 18.15 ng/µL, P=0.0020), higher mean purity (OD260/280 of 1.96 versus 1.70, P=0.0156) and lower mean CT for GAPDH (22.68 versus 29.60, P=0.0020). The authors report that GAPDH was not amplified in the absence of the reverse transcription step, indicating the removal of contaminating DNA was successful in all specimens. Further, the authors report that the miRNA RNA43 was successfully amplified in all specimens.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • Other Preservative
   • PAXgene

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   DNA	Real-time qPCR
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	PAXgene RNApro
   Storage	Storage duration	1 week 1 month 48 h at −80°C 1-6 years at −80°C
   Storage	Storage temperature	-80°C -20°C 4°C Room temperature
   Biospecimen Preservation	RNA stabilization method	RNApro PAXgene tube

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