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Influence of next-generation sequencing and storage conditions on miRNA patterns generated from PAXgene blood.

Author(s): Backes C, Leidinger P, Altmann G, Wuerstle M, Meder B, Galata V, Mueller SC, Sickert D, Stähler C, Meese E, Keller A

Publication: Anal Chem, 2015, Vol. 87, Page 8910-6

PubMed ID: 26207298 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of freeze-thaw cycling and refrigerated versus frozen storage of blood in PAXgene tubes on the miRNA profile. The authors also investigated the effect of NGS preprocessing method on the miRNA profile. Blood was collected from three healthy individuals into five PAXgene tubes and stored at -80°C for 4 days with no freeze/thaw cycling, a freeze/thaw cycle on day 1, a freeze/thaw cycle on days 1 and 2, or a freeze/thaw cycle on days 1, 2, and 3. The final tube was stored at 8°C for the full 4 days. Blood was collected from a fourth healthy individual into five tubes stored at -80°C and used for comparisons between NGS preprocessing methods. miRNA was isolated from all specimens using the PAXgene Blood miRNA Kit. NGS libraries were constructed using the Illumina TruSeq small RNA Library Prep Kit and sequenced using an Illumina HiSeq2500.  The authors state two different preprocessing methods were used but details were not provided.

   Summary of Findings:

   A total of 1252 miRNAs were identified in the blood specimens 1060 of which were found in more than one read and 455 were found in 50 or more reads across the 25 specimens. However, 90.3% of reads mapped to miR-486-5p and 5% to miR-92a-3p, leaving only 4.7% of reads for the other 1250 miRNAs. Read count decreased after the first freeze/thaw cycle and was comparable in specimens subjected to three freeze/thaw cycles and those stored at 8°C. Applying principle component analysis to the 455 miRNAs with more than 50 reads did not show clustering based on patient but instead based on the handling. Specimens that were immediately frozen clustered with the specimens thawed once and one of the specimens thawed twice. The remaining specimens thawed twice and the specimens thawed three times clustered with the refrigerated specimens. Further analysis identified 41 miRNAs significantly affected by storage condition but, after correction for multiple testing, significance was only established for five miRNAs: miR-320b (P = 0.0002), miR-320a (P = 0.001), miR-16-5p (P=0.018), miR-18b-5p (P=0.037), and miR-375 (P=0.0375). As an example, the authors show significantly higher levels of miR-375 in specimens refrigerated for four days versus those stored frozen regardless of freeze/thaw cycling. For most miRNA, the inter-individual variability was greater than that due to differences in specimen handling. In specimens collected from the same patient and handled identically (frozen without freeze/thaw cycling), the miRNA profile clustered strongly based on NGS library preparation method (details not provided). The CV in miRNA expression between library preparation methods for a single individual was lower than that between specimen handling procedures. Only two miRNAs (miR-182−5p and miR-1271−5p) with counts >5 had a CV >1.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • PAXgene
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	-80°C 8°C
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles

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