Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Molecular and pathological evaluation of cryopreserved colorectal cancerous tissues: Effects of freezing method and cryoprotection.

Author(s): Liang W, Ding F, Wang MX, Liu BL, Sun M

Publication: Cryo Letters, 2017, Vol. 38, Page 321-329

PubMed ID: 29734434 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the effects of four different preservation methods on gene expression profiles and histopathology assessment of colorectal cancer tissue specimens. Tumor tissue collected from three colorectal cancer patients that underwent surgical resection was cut into 3x3x3 mm blocks. Blocks were divided into four groups (2 blocks per group) based on preservation method: 1) stabilization in RNAlater at room temperature (control specimens), 2) snap-freezing and subsequent storage in liquid nitrogen for 7 days, 3) cryoprotectant permeation in CPA solution for 30 min at room temperature (storage conditions not provided), or 4) cryopreservation in a rate-controlled freezer (cooled at 1°C/min to -80°C and then held at -80°C for 1 h) after permeation in CPA solution and subsequent storage in liquid nitrogen for 7 days. Additional case-matched samples were formalin-fixed, paraffin-embedded and H-and-E stained for histopathological analysis. Frozen tissue specimens were rapidly thawed in a 37°C water bath, washed with PBS, and stabilized in RNAlater briefly at room temperature prior to RNA extraction and microarray analysis. RNA was extracted from all specimens using the Takara RNAiso Plus kit, quantified using a bioanalyzer, and then further purified using the RNeasy Mini kit. Purified RNA was assessed by spectrophotometry and a bioanalyzer and quality was determined by RIN, 28S/18S, and absorbance (A260/280). All specimens selected for microarray analysis had RIN> 6, 28S/18S ≥ 0.7, and A260/280 ratio of 1.8-2.1. Microarray analysis was performed with the Affymetrix GeneChip PrimeView Human Gene Expression Array.

   Summary of Findings:

   Snap-freezing of colorectal cancer specimens resulted in the highest number of differentially expressed genes when compared to case-matched specimens stabilized in RNAlater (1381 genes total, 965 upregulated, and 416 downregulated) followed by controlled-rate cryopreserved specimens (1381 total, 815 upregulated, and 245 downregulated) and CPA-permeated specimens (813 total, 538 upregulated, and 274 downregulated). Similarly, snap-frozen specimens also demonstrated more genes with a greater than 2-fold change from RNAlater-preserved specimens (48 genes total, 3 upregulated, and 5 downregulated) followed by controlled-rate cryopreserved specimens (16 total, 6 upregulated, and 10 downregulated) and CPA-permeated specimens (3 genes total, 1 upregulated, and 2 downregulated). Scatter plot analysis of differential expression of genes associated with colorectal cancer as well as the magnitude of fold-change revealed an influence due to preservation method but almost all differences fell below the significant threshold (P<0.5). H-and-E comparisons to FFPE specimens revealed shrinkage of nuclei but clear cell margins in snap-frozen and controlled-rate cryopreserved specimens and less clear nuclei margins with no shrinkage for CPA-permeated specimens; however, the authors state that all specimens were acceptable for histopathologic examination.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin
   • Other Preservative
   • RNAlater
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Morphology	H-and-E microscopy
   RNA	Tissue microarray
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	RNAlater Snap frozen Cryoprotective agent Frozen

   View more