Collection and storage of human blood and adipose for genomic analysis of clinical samples.

Author(s): Cashion AK, Umberger RA, Goodwin SB, Sutter TR

Publication: Res Nurs Health, 2011, Vol. 34, Page 408-18

PubMed ID: 21812005 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of storing leukocytes on LeukoLOCK filters with RNAlater for 1 week at -20 degrees C on RNA quality and transcriptome analysis using two different Affymetrix microarray platforms. Blood from healthy individuals was collected into EDTA vacutainers, and leukocytes were isolated on paired LeukoLOCK filters. RNA was preserved on the leukoLOCK filters by the addition of RNAlater to the filter. One specimen from each of the two patients was stored frozen at -20 degrees C on the LeukoLOCK filters for 1 week before extraction, while the other was extracted after only a short duration (unspecified) at room temperature. RNA was isolated using the LeukoLOCK RNA isolation system.

   Summary of Findings:

   RINs of 8.9-9.0 were obtained from fresh specimens and those that were stored at -20 degrees C for 1 week, and any differences in RNA yield were attributed to the learning curve of the authors. Percent present calls on the HG-U133 plus 2.0 array were 48.3% and 46.7% for unfrozen and frozen specimens, respectively. The authors report a similar number of transcripts were detected using the HG-U133 array and the Gene 1.0 ST array.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - White Cells

   Preservative Types

   • RNAlater
   • Frozen
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	DNA microarray
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh) RNAlater
   DNA microarray Specific	Type of array	HG-U133 array GENE 1.0 ST array
   Storage	Storage duration	0 days 1 week

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2. Study Purpose

   The purpose of this study was to determine the effects of RNA isolation method on the quality of RNA from subcutaneous adipose tissue obtained during renal transplant surgery. Abdominal subcutaneous adipose tissue was placed on ice and transported out of the operating room where it was cut into 10 pieces. The pieces of tissue were individually snap-frozen in liquid nitrogen within 2 minutes of collection and stored at -80 degrees C.

   Summary of Findings:

   RNA yields were comparable between the STAT-60 lipids kit and Trizol with plus the RNeasy kit. While the optical density 260/280 ratios were acceptable using both the STAT-60 lipids kit (1.96-1.97) and Trizol plus the RNeasy kit (2.10), only specimens isolated using the Trizol plus the RNeasy kit were intact enough to yield RIN values (8.0). The RNA isolated using the Trizol plus RNeasy kit was of sufficient quality for microarray analysis, and expression of the expected genes involved in lipid metabolism was confirmed.

   Biospecimens

   • Tissue - Adipose

   Preservative Types

   • Frozen

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	STAT-60 for lipids Trizol with RNeasy
   Preaquisition	Diagnosis/ patient condition	Renal transplant recipient

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