背景:随着单细胞测序技术的快速发展,组织学研究不再局限于传统的匀浆化组织。激光显微切割能够准确分离特定的组织或细胞,当与下一代测序相结合时,它可以在细胞水平揭示重要的生物过程。然而,传统的激光显微切割技术往往既复杂又耗时,从收集的样本中提取的RNA的质量不一致,限制后续研究。因此,一个改进的,简单,迫切需要高效的激光显微切割方法。
结果:我们省略了样品固定和冷冻保护剂添加步骤。相反,将新鲜样品包埋在1.5ml离心管盖内的最佳切割温度介质中,用液氮快速冷冻,并立即进行冷冻切片。对柑橘皮的一系列切片厚度进行了RNA提取测试,这表明18μm厚度产生最高质量的RNA。通过将脱水时间缩短至每个乙醇梯度1分钟,并省略组织清除步骤,所得的高效脱水和保存的形态确保了高质量的RNA提取。我们还通过将激光功率调整到最佳值,提出了一套激光显微切割参数,减小光圈大小,降低脉冲频率.收集柑橘皮的表皮和表皮下细胞,RNA提取在9小时内完成。使用这种有效的方法,分离组织的转录组测序产生了高质量数据,平均Q30值和作图率超过91%.此外,转录组分析揭示了细胞层之间的显著差异,进一步证实了我们隔离方法的有效性。
结论:我们开发了一种简单,快速的激光显微切割方法,并通过基于柑橘皮的研究证明了其有效性,我们从中产生高质量的转录组数据。这种快速有效的细胞分离方法,与转录组测序相结合,不仅有助于柑橘细胞水平的精确组织学研究,而且为更广泛的其他植物组织中的细胞特异性转录组分析提供了一种有前途的方法。
BACKGROUND: With the rapid development of single-cell sequencing technology, histological studies are no longer limited to conventional homogenized tissues. Laser microdissection enables the accurate isolation of specific tissues or cells, and when combined with next-generation sequencing, it can reveal important biological processes at the cellular level. However, traditional laser microdissection techniques have often been complicated and time-consuming, and the quality of the RNA extracted from the collected samples has been inconsistent, limiting follow-up studies. Therefore, an improved, simple, and efficient laser microdissection method is urgently needed.
RESULTS: We omitted the sample fixation and cryoprotectant addition steps. Instead, fresh samples were embedded in Optimal Cutting Temperature medium within 1.5 ml centrifuge tube caps, rapidly frozen with liquid nitrogen, and immediately subjected to cryosectioning. A series of section thicknesses of citrus rind were tested for RNA extraction, which showed that 18 μm thickness yielded the highest quality RNA. By shortening the dehydration time to one minute per ethanol gradient and omitting the tissue clearing step, the resulting efficient dehydration and preserved morphology ensured high-quality RNA extraction. We also propose a set of laser microdissection parameters by adjusting the laser power to optimal values, reducing the aperture size, and lowering the pulse frequency. Both the epidermal and subepidermal cells from the citrus rind were collected, and RNA extraction was completed within nine hours. Using this efficient method, the transcriptome sequencing of the isolated tissues generated high-quality data with average Q30 values and mapping rates exceeding 91%. Moreover, the transcriptome analysis revealed significant differences between the cell layers, further confirming the effectiveness of our isolation approach.
CONCLUSIONS: We developed a simple and rapid laser microdissection method and demonstrated its effectiveness through a study based on citrus rind, from which we generated high-quality transcriptomic data. This fast and efficient method of cell isolation, combined with transcriptome sequencing not only contributes to precise histological studies at the cellular level in citrus but also provides a promising approach for cell-specific transcriptome analysis in a broader range of other plant tissues.