Renal tissue thawed for 30 minutes is still suitable for gene expression analysis
Yi Ma1, Xiao-Nan Kang2, Wen-Bin Ding2
1Department 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.
Plos One
|April 2, 2014
Summary
Thawing mouse renal tissue at room temperature for 30 minutes minimally impacts gene expression analysis. Proper normalization using reference genes like B2M, ACTB, and PPIA minimizes errors from RNA degradation.
Area of Science:
- Molecular Biology
- Genomics
- Biobanking
Background:
- Biosamples from biobanks often require thawing before processing.
- Understanding the impact of thawing on gene expression is crucial for accurate analysis.
Purpose of the Study:
- To evaluate the effects of room temperature thawing duration on gene expression profiling.
- To identify optimal reference genes for quantitative real-time polymerase chain reaction (qPCR) normalization in thawed samples.
Main Methods:
- Time-course gene expression profiling using microarrays on thawed mouse renal tissue.
- Validation of microarray data using qPCR on candidate reference and target genes.
- Application of geNorm plus and NormFinder for reference gene stability analysis.
Main Results:
- RNA degradation increased with longer thawing times at room temperature.
- Microarray analysis revealed significant alteration in only 0.91% of genes.
- A combination of B2M, ACTB, and PPIA was identified as the most stable reference gene set for qPCR normalization.
Conclusions:
- Thirty minutes of thawing at room temperature has a limited impact on microarray and qPCR results.
- Proper normalization with validated reference genes effectively reduces gene expression variations due to early RNA degradation.
- Inappropriate reference gene selection can lead to significant gene quantification errors.
