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Comparison of whole blood RNA preservation tubes and novel generation RNA extraction kits for analysis of mRNA and MiRNA profiles.

Author(s): Häntzsch M, Tolios A, Beutner F, Nagel D, Thiery J, Teupser D, Holdt LM

Publication: PLoS One, 2014, Vol. 9, Page e113298

PubMed ID: 25469788 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effect of blood stabilization tube types (PAXgene and Tempus), RNA extraction methods, and reverse-transcription methods on the RNA yield and integrity and levels of three mRNAs and three miRNAs  in blood from patients with and without acute MI. Blood was collected into three PAXgene Blood RNA Tubes and three Tempus Blood RNA Tubes from 12 patients with MI and 35 patients without MI (control). Tubes were stored at room temperature for 2 h, followed by storage at -20°C for 24 h, and then -80°C for more than a week. Blood was thawed for 2 h at room temperature (PAXgene tubes) or 30 min at room temperature (Tempus tubes) before RNA extraction. RNA was extracted from blood in PAXgene tubes using  the PAXgene Blood miRNA Kit, the Norgen Preserved Blood RNA Purification Kit II, or the QIAsymphony PAXgene Blood RNA Kit for fully automated RNA isolation on the QIAsymphony and extracted from blood in Tempus tubes using MagMAX for Stabilized Blood Tubes RNA Isolation Kit, Norgen Preserved Blood RNA Purification Kit I, or the  MagMAX for Stabilized Blood Tubes RNA Isolation Kit for semi-automated isolation on the MagMAX Express-96 Magnetic Particle Processor. RNA concentration was assessed by spectrophotometry. RNA was frozen at -80°C and then quality was assessed using the Agilent Small RNA Kit on a Bioanalyzer 2100. RNA from 12 specimens (6 MI and 6 control) extracted using the Qiagen kits and Life Technologies kits was reverse-transcribed and quantified in the following ways: (1) reverse-transcribed with the miRScript Reverse Transcription Kit and mRNA quantified using QuantiTect Primer assays (mRNA), (2) reverse-transcribed with the miRScript Reverse Transcription Kit and miRNA quantified using the miScript Primer assay (miRNA), (3) reverse-transcribed with Supercripts Vilo cDNA Synthesis Kit and mRNA quantification using TaqMan assays, or (4) reverse-transcribed using TaqMan Small RNA assays and quantification using TaqMan miRNA assays. All cDNA was stored at -20°C until quantification.

   Summary of Findings:

   For PAXgene specimens, significantly more RNA per g of blood was obtained using the QIAsymphony and PAXgene kits than the Norgen kit in patients with (P<10^–5) and without MI (P<10^–5), but there were no significant differences in RNA yield among kits for Tempus specimens. Significantly more RNA per g blood was obtained from specimens in Tempus tubes than PAXgene tubes in the control group (P=3.54 x 10^-4) but no difference was found in the MI group. More RNA per g of blood was also obtained from the blood of patients in the MI group than the control group (P=5.3 x 10^-9), regardless of tube type. Similar effects were found when yield of RNA was not normalized to input. RIN values were comparable among extraction methods but tended to be lower when RNA was extracted with the Norgen kits rather than the other methods (5.9-6.0 versus 6.0-8.6). RNA extracted from PAXgene blood using the Norgen Kit had higher CT values for ACTB, MMP9, and ARG1 and lower CT values for miR-16 and miR30b than RNA from Tempus blood or RNA from PAXgene blood extracted using the other kits (no statistics evaluated). mRNA CT values were higher when reverse-transcription was with Superscript VILO cDNA Synthesis Kit rather than miScript Reverse Transcription Kit. In contrast, there was more variability in miRNA expression levels, with each miRNA affected differently by RNA extraction method used, tube type, and reverse-transcription method. The CV for the three RNA transcripts were all below 1.0 regardless of tube type, extraction method, or reverse-transcription method except for ACTB and MMP9 when RNA was extracted from PAXgene tubes using the Norgen II Kit and reverse-transcribed using miScript. The CVs for miRNAs were also below 1.0 except for miR31 when reverse-transcription was with miScript. MMP9 and ARG1 levels were increased in specimens from MI patients compared to controls, regardless of tube type or extraction and reverse-transcription method. Although more variable in extent, miR133a and miR1 levels were generally higher in MI patients than controls.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • Frozen
   • PAXgene

   Diagnoses:

   • Not specified
   • Cardiovascular Disease

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Acute myocardial infarction No myocardial infarction
   Biospecimen Acquisition	Type of collection container/solution	PAXgene blood RNA tube Tempus blood RNA tube
   Analyte Extraction and Purification	Analyte isolation method	PAXgene Blood miRNA Kit Norgen Preserved Blood RNA Purification Kit II QIAsymphony PAXgene Blood RNA Kit for fully automated RNA isolation MagMAX for Stabilized Blood Tubes RNA Isolation Kit Norgen Preserved Blood RNA Purification Kit I MagMAX for Stabilized Blood Tubes RNA Isolation Kit on the MagMAX Express-96 Magnetic Particle Processor
   Spectrophotometry Specific	Data handling	Not normalized Normalized to g blood
   Real-time qRT-PCR Specific	Technology platform	TaqMan assays QuantiTect Primer assays miScript Primer assay
   Real-time qRT-PCR Specific	Targeted nucleic acid	ACTB MMP9 ARG1 miR1 miR16 miR30b miR133a
   Real-time qRT-PCR Specific	Template modification	Reverse transcribed with miRScript Reverse Transcription kit Reverse transcribed with Supercripts Vilo cDNA Synthesis kit Reverse transcribed using TaqMan small RNA assays

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