RNA analysis based on a small number of manually isolated fixed cells (RNA-snMIFxC) to profile stem cells from human deciduous tooth-derived dental pulp cells.
RNA analysis based on a small number of manually isolated fixed cells (RNA-snMIFxC) to profile stem cells from human deciduous tooth-derived dental pulp cells.
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DOI：
10.1186/s12575-021-00149-5
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发表时间：
2021-06-11
影响因子：
6.4
通讯作者：
Sato M
中科院分区：
文献类型：
--
作者：
Inada E;Saitoh I;Kubota N;Iwase Y;Kiyokawa Y;Noguchi H;Yamasaki Y;Sato M
通过固定和随后的免疫或细胞化学染色，可以评估人乳牙源性牙髓细胞 (HDDPC) 中干性因子的表达，例如八聚体结合转录因子 3/4 (OCT3/4)、性别决定区 Y-box 2 (SOX2) 和碱性磷酸酶 (ALP)。荧光激活细胞分选（FACS）是一种收集感兴趣细胞的强大系统，但受到仪器成本和操作难度的限制。与 FACS 相比，磁激活细胞分选便宜，但仅限于表面表达靶分子的细胞。在这项研究中，开发了一种简单且廉价的方法，通过使用吸嘴控制的微量移液器在解剖显微镜下分离一些细胞，对具有靶分子细胞内表达的免疫或细胞化学染色的细胞进行分子分析。用 3,3'-二氨基联苯胺等显色剂染色后，两个或多个有色细胞 (~ 10) 成功地与未染色的细胞分离。 3-磷酸甘油醛脱氢酶（一种管家基因）的表达在所有样本中均可辨别，而干性基因（例如 OCT3/4、SOX2 和 ALP）的表达仅限于阳性染色的细胞。这些发现表明这些方法在小规模细胞分析中表现出干性特异性基因产物的细胞质或核定位的保真度。
Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette. Two or more colored cells (~ 10), after staining with a chromogen such a 3,3′-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells. These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.
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影响因子：
4.1
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