| Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs
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Author:
Date:
2020-10-05
[Abstract] Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA’s intracellular abundance, i.e., approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of interest. At least two approaches have been used to approximate lncRNA intracellular abundance: single-molecule sensitivity RNA fluorescence in situ hybridization (smFISH) and single-gene, calibrated reverse-transcription followed by quantitative PCR (RT-qPCR). However, like all experimental approaches, these methods have their limitations. smFISH, when analyzed using diffraction-limited microscopy, can underestimate intracellular ...
[摘要] [摘要]长非编码RNA（lncRNA）在正常生理和疾病中起着至关重要的作用，但其作用机理可能难以鉴定。对于机理研究，了解lncRNA的胞内丰度（即在目标细胞类型的典型细胞中大约存在多少个lncRNA分子）通常很有用。至少两种方法已用于估算lncRNA细胞内丰度：单分子敏感性RNA荧光原位杂交（smFISH ...
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| Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue
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Author:
Date:
2020-09-20
[Abstract] Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available. We therefore developed Probe-Seq, which uses hybridization of gene-specific probes to RNA markers for isolation of specific types of cells, to enable downstream FACS isolation and bulk RNA sequencing. We show that this method can enable isolation and profiling of specific cell types from mouse retina, frozen human ...
[摘要] [摘要 ] 大多数器官和组织由许多类型的细胞组成。为了表征细胞状态，目前可以使用各种转录分析方法，包括全组织体RNA测序，单细胞RNA测序（scRNA -Seq）和特定于细胞类型的RNA测序。在此库中缺少的是一种简单，通用的方法，无法批量获得细胞类型的特定基因标记或抗体，因此无法批量转录。因此，我们开发了Probe-Seq，该探针使用基因特异性探针与RNA标记的杂交来分离特定类型的细胞，以实现下游FACS分离和大量RNA测序。我们表明，该方法可以实现从小鼠视网膜，冷冻人视网膜，果蝇中肠和发育中的雏鸡视网膜中分离和分析特定细胞类型的特征，这表明它对大多数生物很有用。
[背景技术 [ 0002 ] 在过去的二十年中，使用RNA-Seq和微阵列进行转录谱分析已在生物学研究中无处不在。分析现在是用来了解大多数生物体中细胞和细胞状态的主要工具之一。它被用于正常发育，异常发育和疾病的研究，并极大地扩展了我们对进化关系的理解。特别地，scRNA- Seq已经以前所未有的速度导致了新型细胞类型的鉴定（Picelli 等，2013；Jaitin 等，2014； Klein 等，2015； Macosko 等，2015）。为了更深入地了解这些新描述的细胞类型，一种无需转基因标记或特异性抗原即可将其分离的方法将大有裨益。尽管可以使用scRNA ...
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| Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism
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Author:
Date:
2018-09-05
[Abstract] Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery.
The ...
[摘要] 使用气相色谱质谱（GC-MS）分析柠檬酸循环（CAC）和相关中间体（如谷氨酸，谷氨酰胺，GABA和天冬氨酸）是一种分析方法，用于识别明显相关和不相关的能量途径之间的意外相关性代谢。通过使用已知量的样品添加的参考标准，中间体可以表示为它们的绝对浓度或相对比例。 GC-MS还可以区分重标记分子（ 2 H-或 13 C-标记的）和天然存在的最丰富的分子。使用示踪剂的应用还可以评估在各种生理和病理条件下以及用于途径发现的特定代谢池的周转。
以下方案是一种相对简单的方法，不仅对小浓度的代谢中间体敏感，而且还可以 in vivo 或 in vitro 用于确定各种新陈代谢的完整性途径，如特定代谢物池内的通量变化。我们使用该协议来确定磷酸烯醇丙酮酸羧激酶1 （ Pck1 ）基因在小鼠巨噬细胞中的作用，以确定 13 C将葡萄糖标记为特定的CAC代谢物库
【背景】随着细胞和小鼠中基因表达改变的发展，需要了解这些缺失或过表达的基因如何影响代谢途径的调节。在该方案中，我们使用稳定同位素来确定进入CAC的葡萄糖通量如何改变葡萄糖对柠檬酸盐，琥珀酸盐和苹果酸盐的贡献。使用稳定同位素和目标分析代谢只是在细胞培养中使用稳定同位素的一个好处。
本方案中描述的用于细胞内代谢物功能定量的方法是通过在U- 13 ...
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