Effects of tissue handling on RNA integrity and microarray measurements from resected breast cancers.

Author(s): Hatzis C, Sun H, Yao H, Hubbard RE, Meric-Bernstam F, Babiera GV, Wu Y, Pusztai L, Symmans WF

Publication: J Natl Cancer Inst, 2011, Vol. 103, Page 1871-83

PubMed ID: 22034635 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   In order to assess the impact cold ischemia time and preservation method have on RNA yield, integrity, and expression, breast tumor specimens from 17 patients were minced, stirred, and divided into 8 equal portions that were then subjected to 0, 20, 40, 60, 120, or 180 min at room temperature if preserved by RNAlater or 0 or 40 min if preserved by snap-freezing on dry ice. Importantly, the cold ischemia time associated with pathological evaluation was recorded and ranged between 23 and 68 min, but was not included in the author-specified cold ischemia timepoints. All specimens were stored at -80 degrees C until analysis, and RNAlater-preserved specimens were held at room temperature for approximately 60 min prior to freezing.

   Summary of Findings:

   With regard to preservation method, RNAlater-preserved specimens had greater RNA yields (by 3-fold) and higher RIN (8.13 versus 7.31) compared to case-matched specimens snap frozen on dry ice. Of the 86 microarrays constructed from 11 patients, 5% failed microarray quality control tests although 8% had RIN values less than 6. Differences between preservation methods included a significantly lower 3'/5' ratio in RNAlater-preserved specimens compared to snap frozen specimens for beta-actin, gyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S rRNA (p<0.001) and signal transducer and activator of transcription 1 (STAT1; p=0.014), but lower average ER (p=0.003) and HER-2 (p=0.016) signals in RNAlater preserved specimens. When cold ischemia time was investigated, neither RNA yield nor RIN were affected by 40 min of cold ischemia compared to 0 min controls in snap frozen specimens. For RNAlater-preserved specimens, prolonged cold ischemia resulted in modest declines in RNA yield (1.5 ug/h; p=0.19) and RIN (0.12 unit/h; p=.008), while levels of single mRNA targets and multigene indices remained stable with the exception of a modest but significant decrease in GGI (p=0.007, -5.4 units/h). However, regression analysis revealed that 3'/5' ratios increased significantly for STAT1 (p=0.001), beta-actin (p=0.02), and 18S rRNA (p=0.006), and decreased for 28S rRNA (p=0.006) following prolonged cold ischemia in specimens preserved in RNAlater. RNA yield, but not RIN, was significantly affected by surgery type and tumor size (p=0.001). Warm ischemia time and the cold ischemia time associated with pathological evaluation did not significantly affect microarray results, although data was not shown.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • RNAlater
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   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	RNAlater Snap frozen
   Preaquisition	Warm ischemia time	12-118 min
   Biospecimen Acquisition	Cold ischemia time	0 min 20 min 40 min 60 min 120 min 180 min
   DNA microarray Specific	Targeted nucleic acid	ER HER-2 ACTB STAT1 18S rRNA 28S rRNA
   Biospecimen Acquisition	Method of tissue acquisition	Mastectomy Segmental mastectomy

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