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首页> 外文期刊>Cell and tissue banking: An international journal of banking, engineering & transplantation of cells and tissues >Cryopreserved Human B Cells as an Alternative Source for Single Cell mRNA Analysis
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Cryopreserved Human B Cells as an Alternative Source for Single Cell mRNA Analysis

机译：冷冻保存的人类B细胞作为单细胞mRNA分析的替代来源

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摘要

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO] – stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the ‘house-keeping gene’ glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B?cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells.
机译：单个B淋巴细胞的逆转录聚合酶链反应（RT-PCR）已被证明是同时分析恶性和非恶性B细胞亚群中不同mRNA特异性的强大工具。但是，特别是对于纵向研究，这可能还需要分析冷冻保存的细胞。因此，当前的研究评估了冷冻保存（液氮，二甲基亚砜[DMSO] –储存）的存活B细胞是否是单细胞RT-PCR分析的替代来源。通过荧光激活细胞分选术（FACS）分析新鲜（非冷冻）和解冻后的人外周血B细胞。结果，尽管在分析冷冻保存的细胞时观察到某些亚群标记的荧光强度略有减弱，但通过四色流式细胞术可以可靠地将不同的B细胞亚群染色并与新鲜和解冻后的细胞分离。随后，将可存活的单个CD19 + CD27 +记忆B细胞分类到单个孔中，并分析“看家基因”甘油醛磷酸脱氢酶（GAPD），组成型B细胞归巢受体CXCR4和免疫球蛋白重链的mRNA转录表达。巢式RT-PCR方案检测可变区（IgVH）基因。比较这两种B细胞来源，RT-PCR分析显示了三种测试的mRNA特异性转录本（GAPD，CXCR4，IgVH）表达转录本的细胞的产量相当，表明冷冻保存的Bα细胞中各个mRNA的完整性。总之，这些数据表明，最佳冷冻保存的B细胞可能是单细胞RT-PCR分析的替代来源，尤其是在纵向B细胞研究中。但是，在分析解冻后的细胞时，应针对每个不同的亚群和感兴趣的靶标mRNA重新评估FACS分析和RT-PCR的设置。

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