Impact of collection and storage of lung tumor tissue on whole genome expression profiling.

Author(s): Freidin MB, Bhudia N, Lim E, Nicholson AG, Cookson WO, Moffatt MF

Publication: J Mol Diagn, 2012, Vol. 14, Page 140-8

PubMed ID: 22240448 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 warm and cold ischemia time, preservation method, and frozen storage on the mRNA quality from lung cancer specimens. Specimen were collected from 6 patients at chest opening and immediately after resection and placed in RNAlater for 24 h at 4 degrees C before storage at -80 degrees C. An additional 4 specimens were collected from each of 6 patients after transfer to histopatholgy (~30 min cold ischemia) and were either placed in RNAlater at 4 degrees C for 24 h and stored at -80 degrees C, fixed in formol saline for 24 h and embedded in paraffin, snap-frozen and processed immediately, or snap-frozen and stored for 1 week at -80 degrees C. RNA was extracted from RNAlater specimens using the RNeasy fibrous tissue minikit, but was extracted from formol saline-fixed paraffin embedded tissue sections using the RecoverAll total nucleic acid kit.

   Summary of Findings:

   The authors report that the yield of RNA was independent of collection time and preservation method. RNA obtained from specimens collected into RNAlater at chest opening, after lung resection, or after transport to histopathology had the highest mean RNA integrity numbers RIN (7.4, 7.7 and 7.9, respectively). On the other hand, RNA obtained from specimens that were snap-frozen after transfer to histopathology and thawed prior to extraction was more degraded with or without 1 week of storage at -80 degrees C (median RIN 5.2 and 4.4, respectively). Finally, the RIN from formol saline-fixed paraffin embedded specimens ranged from 2.2-2.5.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • RNAlater
   • Frozen
   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	0 min 30 min
   Biospecimen Preservation	Type of fixation/preservation	Formol saline RNAlater Snap frozen
   Storage	Storage duration	0 days 1 week
   Preaquisition	Warm ischemia time	0 min Unspecified

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2. Study Purpose

   The purpose of this study was to determine the effects of warm and cold ischemia time, preservation method, and frozen storage on the gene expression profile from lung cancer specimens. Specimen were collected from 6 patients at chest opening and immediately after resection and placed in RNAlater for 24 h at 4 degrees C before storage at -80 degrees C. An additional 4 specimens were collected from each of 6 patients after transfer to histopatholgy (~30 min cold ischemia) and were either placed in RNAlater at 4 degrees C for 24 h and stored at -80 degrees C, fixed in formol saline for 24 h and embedded in paraffin, snap-frozen and processed immediately, or snap-frozen and stored for 1 week at -80 degrees C. RNA was extracted from RNAlater specimens using the RNeasy fibrous tissue minikit, but was extracted from formol saline-fixed paraffin embedded tissue sections using the RecoverAll total nucleic acid kit. RNA was hybridized to Illumina Human WG-6 arrays.

   Summary of Findings:

   While 12,000-13,000 probes were detected using RNA from the snap-frozen and RNAlater preserved specimens, only 3000 probes had signals above background using formol saline-fixed paraffin embedded specimens. The authors report the highest signal to background ratios were generated using the specimens collected into RNAlater prior to transport to histopathology, and the lowest signal to background ratios were generated using RNA from formol saline-fixed paraffin embedded specimens. Only 5% of genes were differentially expressed between specimens collected at chest opening and those collected after surgical resection (warm ischemia time), and only 1% differed by more than 2-fold. Pathways analysis revealed that that inflammation and wound healing pathways were significantly affected by surgical resection. While 23.5-37% of genes, including 68 of 119 genes previously suggested as lung cancer biomarkers, differed in expression between specimens collected before transport to histopathology (at the time of chest opening or surgical resection) and after transport to histopathology (cold ischemia time), only 0.2-0.4% of genes were differentially expressed between specimens collected after transport and either preserved with RNAlater or snap-frozen. Further, specimens collected before transport clustered together as did the frozen and RNAlater preserved specimens collected after transport. Pathway analysis showed the pathways differentially expressed between specimens collected before and after transport were predominantly biopolymer and macromolecule metabolism. In contrast, 89.4-91.6% of genes were differentially expressed between formol saline-fixed paraffin embedded specimens and the RNAlater specimens collected at chest opening, and the correlation of expression was only r2=0.142.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • RNAlater
   • Frozen
   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	0 min 30 min
   Biospecimen Preservation	Type of fixation/preservation	Formol saline RNAlater Snap frozen
   Storage	Storage duration	0 days 1 week
   Preaquisition	Warm ischemia time	0 min Unspecified

   View more