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Longitudinal assessment and stability of long non-coding RNA gene expression profiles measured in human peripheral whole blood collected into PAXgene blood RNA tubes.

Author(s): Wylezinski LS, Shaginurova GI, Spurlock Iii CF

Publication: BMC Res Notes, 2020, Vol. 13, Page 531

PubMed ID: 33183338 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of storing PAXgene blood, extracted RNA, and cDNA at -80°C for up to 1 year on RNA yield and integrity as well as levels of six lncRNAs. The effects of freeze/thaw cycling extracted RNA or cDNA on levels of six lncRNAs and six mRNAs were also investigated. Blood was collected from five healthy patients into multiple PAXgene Blood RNA tubes, pooled, and aliquoted into new PAXgene tubes. PAXgene tubes were stored at -80°C for up to 1 year (monthly timepoints) or used for immediate RNA extraction. RNA was isolated using a QIAcube system and further purified using the RNEasy MinElute Cleanup Kit. To investigate the effects of storage, RNA extracted from fresh specimens was used immediately or stored for a year at -80°C prior to cDNA synthesis. To investigate the effects of freeze/thaw cycling of RNA, some specimens were thawed at room temperature for 10 min and refrozen 0, 5, or 10 times at -80°C before reverse transcription. cDNA was synthesized using Superscript III First-Strand Synthesis System with oligo-dT priming. To investigate the effects of storage of cDNA, some specimens were used immediately while others were stored for a year at -80°C or freeze/thaw cycled 0, 5, or 10 times prior to lncRNA quantification. RNA and cDNA were quantified by spectrophotometer. Integrity was evaluated by bioanalyzer. lncRNAs were quantified by custom real-time RT-PCR assays and normalized to GAPDH.

   Summary of Findings:

   Comparable RNA integrity numbers (RINs) and levels of total RNA and of the six lncRNAs investigated were obtained when RNA was extracted and used for real-time RT-PCR without any storage and when PAXgene blood was stored for one year at -80°C before extraction and RT-PCR. However, over the course of the year of storage, significant differences in the 95% confidence interval from baseline were found in Month 10 for RP11-125,214.2, AC012314.8, and RP11-335I12.2 and in Months 2, 4, 6, and 7 for MCCC1-AS1. Importantly, the 95% confidence intervals overlapped when the months were compared for all lncRNAs except MCCC1-AS1. Levels of the six lncRNAs were comparable in specimens stored for a year at -80°C as extracted RNA or as cDNA to those in specimens used for real-time RT-PCR immediately after RNA extraction. RINs were comparable in RNA subjected to 0, 5, or 10 freeze/thaw cycles. Additionally, levels of six lncRNAs and six mRNAs were unaffected by freeze/thaw cycling of the RNA or cDNA up to 10 times.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 months 1 months 2 months 3 months 4 months 5 months 6 months 7 months 8 months 9 months 10 months 11 months 12 months
   Real-time qRT-PCR Specific	Targeted nucleic acid	RP11-97C16.1 LINC00847 RP11-125,214 2AC012314.8 RP11-335I12.2 MCCC1-AS1
   Storage	Freeze/thaw cycling	0 cycles 5 cycles 10 cycles
   Storage	Storage conditions	As blood As extracted RNA As cDNA

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