Renal tissue thawed for 30 minutes is still suitable for gene expression analysis

Yi Ma; Xiao-Nan Kang; Wen-Bin Ding; Hao-Zheng Yang; Ye Wang; Jin Zhang; Yi-Ran Huang; Hui-Li Dai

doi:10.1371/journal.pone.0093175

Renal tissue thawed for 30 minutes is still suitable for gene expression analysis

PLoS One. 2014 Mar 31;9(3):e93175. doi: 10.1371/journal.pone.0093175. eCollection 2014.

Authors

Yi Ma¹, Xiao-Nan Kang², Wen-Bin Ding², Hao-Zheng Yang³, Ye Wang², Jin Zhang⁴, Yi-Ran Huang⁴, Hui-Li Dai²

Affiliations

¹ 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.
² Department of Biobank, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
³ Department of Central Laboratory, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
⁴ Department of Urology, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Gene Expression / genetics*
Gene Expression Profiling / methods
Kidney
Mice
Mice, Inbred C57BL
RNA / genetics
RNA Stability / genetics
Reference Standards
Specimen Handling / methods*
Temperature
Tissue Array Analysis / methods

Substances

RNA

Grants and funding

This work was supported by Science and Technology Commission of Shanghai Municipality (124119a1300; 12DZ2295005) and Shanghai JiaoTong University School of Medicine (12XJ10014; YBKL2013004). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.