circRNA expression analysis in lung adenocarcinoma: comparison of paired fresh frozen and formalin-fixed paraffin-embedded specimens.
Author(s): Zhang F, Zhao X, Dong H, Xu J
Publication: Biochem Biophys Res Commun, 2018, Vol. 500, Page 738-743
PubMed ID: 29679573 PubMed Review Paper? No
Purpose of Paper
This paper compared expression of circular RNA (circRNA) in formalin-fixed paraffin-embedded (FFPE) specimens with those snap-frozen in liquid nitrogen. Profiles of circRNA expression were examined by microarray and qRT-PCR in case-matched lung adenocarcinoma and normal adjacent specimens procured from three patients.
Conclusion of Paper
CircRNA expression profiles generated with FFPE and snap-frozen lung specimens were modestly to strongly and significantly correlated among both lung adenocarcinoma and normal adjacent specimens examined by microarray. The number of circRNAs that were differentially expressed between tumor and normal adjacent lung specimens was 3,458 when specimens were snap-frozen, and 1,099 for FFPE specimens. Three hundred circRNAs differentially expressed between tumor and normal adjacent specimens were common among FFPE and snap-frozen specimens. Of the four circRNAs that were confirmed by qRT-PCR, three displayed consistent findings with microarray data and included both up- and down-regulated expression in tumor specimens compared to normal adjacent specimens. The authors also reported that the arraystar and CircInteractome screening programs generated predicted interactions for the three circRNAs that displayed differential expression between tumor and normal adjacent specimens by both microarray and qRT-PCR.
Studies
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Study Purpose
This study compared expression of circular RNA (circRNA) in lung adenocarcinoma and normal adjacent specimens procured from three male patients that were preserved by formalin fixation and paraffin embedding to those that were snap frozen in liquid nitrogen. All specimens were preserved within 30 min of surgical excision. Snap-frozen specimens were frozen in liquid nitrogen while FFPE specimens were fixed in 10% formalin for 24 to 48 h. FFPE blocks were stored at room temperature in a dry location for an unspecified duration prior to analysis. Total RNA was isolated from both FFPE and snap-frozen specimens using the Trizol method. RNA purity and concentration were determined by NanoDrop spectrophotometry. Isolated circRNA was transcribed using random hexamer primers and hybridized to Arraystar Human circRNA Arrays V2 and data was obtained with an Agilent Scanner and software. circRNA expression data generated by microarray was verified by qRT-PCR using the 2-∆∆Ct method using levels of beta-actin as a normalizer.
Summary of Findings:
CircRNA expression profiles generated with FFPE and snap-frozen lung specimens were modestly to strongly and significantly correlated among both adenocarcinoma (r= 0.543 to 0.727; p< 0.001) and normal adjacent specimens (r=0.661 to 0.769 ; p< 0.001)when examined by microarray. The number of circRNAs that were differentially expressed between tumor and normal adjacent lung specimens was 3,458 when snap-frozen specimens were compared, and 1,099 when FFPE specimens were compared. Combination of the two data sets revealed 300 circRNAs that were differentially expressed between tumor and normal adjacent specimens among both FFPE and snap-frozen specimens, four of which were further investigated by qRT-PCR. Of those four circRNAs, three displayed consistent findings with microarray data; in tumor specimens two circRNAs were upregulated by 4.31- and 1.48-fold, respectively (hsa_circRNA_100421, hsa_circRNA_100329) and one (hsa_circRNA_101969) was downregulated by 1.49-fold. The authors stated that qRT-PCR results obtained for the remaining circRNA (hsa_circRNA_407081) did not match those generated by microarray. The authors also reported that the arraystar and CircInteractome screening programs generated predicted interactions for the three circRNAs that displayed differential expression between tumor and normal adjacent specimens by both microarray and qRT-PCR.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
Real-time qRT-PCR Specific Targeted nucleic acid hsa_circRNA_100421
hsa_circRNA_100329
hsa_circRNA_101969
hsa_circRNA_407081
beta-actin
Preaquisition Diagnosis/ patient condition Tumor
Normal adjacent