Effects of brain tissue section processing and storage time on gene expression.

The pre-processing of samples is important factors that affect the results of the RNA-sequencing (RNA-seq) data. However, the effects of frozen sections storage conditions on the integrity of RNA and sequencing results haven't been reported. The study of frozen section protection schemes can provide reliable experimental results for single-cell and spatial transcriptome sequencing. In this study, RNA was isolated to be studied for RNA from brain section at different temperatures (RT: room temperature, -20 °C) and storage time (0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 24 h, 7day, 3week, 6month). The stability of reference genes was validated using reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that the storage at room temperature significantly affected RNA integrity number (RIN), and the RIN value was lower with the prolongation of storage, while the storage at -20 °C exerted less effect on the RIN value. Cresyl violet staining for brain tissue sections had little effect on RNA integrity. 1925, 899 and 3390 differential expression genes (DEGs) were screened at 2 h, 4 h and 8 h at room temperature, respectively. A total of 892, 478 and 619 genes were shown to be differentially expressed at -20 °C for 7d, 3w and 6 m, respectively. Among them, the expression of glycoprotein m6a (Gpm6a), calmodulin 1 (Calm1), calmodulin 1 (Calm2), thymosin, beta 4, X chromosome (Tmsb4x), ribosomal protein S21 (Rps21) and so on were correlated with RNA quality. According to the expression stability of 4 reference genes (Actb: beta-actin; Gapdh: glyceraldehyde-3-phosphate dehydrogenase; 18S: 18S ribosomal; Hprt1: hypoxanthine phosphoribosyltransferase 1), 18S is the most stable reference gene in the brain. In conclusion, the storage temperature and time of frozen sections have significant effects on RNA integrity and sequencing results. But there are still some genes that are stable and not affected by worsening of overall RNA integrity ie the decrease of RIN value. In addition, 1% cresyl violet staining can protect RNA.