title: Renal tissue thawed for 30 minutes is still suitable for gene expression analysis.
abstract: Some biosamples obtained from biobanks may go through thawing before processing. We aim to evaluate the effects of thawing at room temperature for different time periods on gene expression analysis. A time course study with four time points was conducted to investigate the expression profiling on 10 thawed normal mice renal tissue samples through Affymetrix GeneChip mouse gene 2.0 st array. Microarray results were validated by quantitative real time polymerase chain reactions (qPCR) on 6 candidate reference genes and 11 target genes. Additionally, we used geNorm plus and NormFinder to identify the most stably expressed reference genes over time. The results showed RNA degraded more after longer incubation at room temperature. However, microarray results showed only 240 genes (0.91%) altered significantly in response to thawing at room temperature. The signal of majority altered probe sets decreased with thawing time, and the crossing point (Cp) values of all candidate reference genes correlated positively with the thawing time (p<0.05). The combination of B2M, ACTB and PPIA was identified as the best choice for qPCR normalization. We found most target genes were stable by using this normalization method. However, serious gene quantification errors were resulted from improper reference genes. In conclusion, thirty minutes of thawing at room temperature has a limited impact on microarray and qPCR analysis, gene expression variations due to RNA degradation in early period after thawing can be largely reduced by proper normalization.
authors.affiliation: Department of Biobank, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China; Department of Urology, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Department of Biobank, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
authors.forename: Xiao-Nan
authors.initials: XN
authors.lastname: Kang
authors.affiliation: Department of Biobank, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Ding
authors.affiliation: Department of Central Laboratory, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
authors.forename: Hao-Zheng
authors.initials: HZ
authors.lastname: Yang
authors.affiliation: Department of Biobank, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Urology, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Urology, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
authors.forename: Yi-Ran
authors.initials: YR
authors.lastname: Huang
authors.affiliation: Department of Biobank, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
authors.forename: Hui-Li
authors.initials: HL
authors.lastname: Dai
mesh_terms: D000818:Animals; D015870:Gene Expression / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods; D007668:Kidney; D051379:Mice; D008810:Mice, Inbred C57BL; D012313:RNA / Q000235:genetics; D020871:RNA Stability / Q000235:genetics; D012015:Reference Standards; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D046888:Tissue Array Analysis / Q000379:methods
chemical_list: D012313:RNA