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.
Highlights
Today biological samples become an increasingly important tool for biomedical research into human diseases [1,2]
Tissue Collection and Processing In order to exclude some pre-analytical factors like anesthesia, operation or transport time in human tissue samples, normal renal tissue was obtained from ten C57BL/6 10-week-old mice and each was immediately sectioned into four aliquots
RNA integrity number (RIN) values of the four pooled samples decreased with thawing time (0 min: 9.5; 5 min: 9.2; 15 min: 8.8; 30 min: 8.0) (Fig. 1), which indicated a measureable RNA degradation in thawed tissue, all the RIN values were in the range of .6 which was recommended as a threshold for high and low quality RNA samples [17]
Summary
Today biological samples become an increasingly important tool for biomedical research into human diseases [1,2]. High quality biosample with RNA closely representing the amount of transcripts in vivo can ensure a more accurate downstream molecular assay [3,4,5]. Some pre-analytical factors like biosample collection, handling, or processing can affect the RNA quality [6,7,8,9]. Some fresh tissue handling protocols proposed that 30 minutes is the longest acceptable time period between surgical removal and freezing [10]. Tissue samples often come from biobanks and may undergo thawing before being processed (e.g. RNA extraction). There is paucity of studies demonstrating the suitable time limit for thawing at room temperature within which DNA, RNA and protein analyses were not impacted
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