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.

中文翻译：

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脑组织切片处理和保存时间对基因表达的影响

样本的预处理是影响 RNA 测序 (RNA-seq) 数据结果的重要因素。然而，冷冻切片储存条件对 RNA 完整性和测序结果的影响尚未见报道。冰冻切片保护方案的研究可为单细胞和空间转录组测序提供可靠的实验结果。在这项研究中，从脑切片中分离 RNA 以研究在不同温度（RT：室温，-20 °C）和储存时间（0 小时、2 小时、4 小时、8 小时、12 小时、16 小时）下的 RNA 、24 小时、7 天、3 周、6 个月）。使用逆转录定量实时聚合酶链反应（qRT-PCR）验证参考基因的稳定性。结果表明，室温储存显着影响RNA完整性数（RIN），并且随着储存时间的延长，RIN值降低，而-20℃的储存对RIN值的影响较小。脑组织切片的甲酚紫染色对 RNA 完整性几乎没有影响。分别在室温下 2 h、4 h 和 8 h 筛选出 1925、899 和 3390 个差异表达基因 (DEG)。共有 892、478 和 619 个基因分别在 -20 °C 7d、3w 和 6 m 时差异表达。其中，糖蛋白m6a（Gpm6a）、钙调蛋白1（Calm1）、钙调蛋白1（Calm2）、胸腺素、β4、X染色体（Tmsb4x）、核糖体蛋白S21（Rps21）等的表达与RNA质量相关。根据4个参考基因的表达稳定性（Actb：β-肌动蛋白；Gapdh：甘油醛-3-磷酸脱氢酶；18S：18S核糖体；Hprt1：次黄嘌呤磷酸核糖基转移酶 1)、18S 是大脑中最稳定的参考基因。总之，冷冻切片的储存温度和时间对RNA完整性和测序结果有显着影响。但仍有一些基因是稳定的，不受整体 RNA 完整性恶化（即 RIN 值降低）的影响。此外，1% 甲酚紫染色可以保护 RNA。