| Extraction and Measurement the Activities of Cytosolic Phosphoenolpyruvate Carboxykinase (PEPCK) and Plastidic NADP-dependent Malic Enzyme (ME) on Tomato (Solanum lycopersicum)
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Author:
Date:
2014-05-05
[Abstract] A recent study demonstrated that cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and NADP-malic enzyme (NADP-ME) have an important role in malate metabolism during fruit ripening (Osorio et al., 2013). PEPCK catalyze the ATP-dependent decarboxylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and NADP-ME, the reversible conversion of malate and pyruvate. Here, we present the detailed protocols to measure PEPCK activity in carboxylation direction by following oxidation of NADH and to measure NADP-ME activity based upon the reduction of NADP+.
[摘要] 最近的研究表明胞质磷酸烯醇丙酮酸羧激酶（PEPCK）和NADP-苹果酸酶（NADP-ME）在果实成熟期间在苹果酸代谢中具有重要作用（Osorio等人，2013）。 PEPCK催化草酰乙酸（OAA）向磷酸烯醇丙酮酸（PEP）和NADP-ME的ATP依赖性脱羧，苹果酸和丙酮酸的可逆转化。 在这里，我们提出详细的协议，以测量PEPCK活性的羧化方向通过以下氧化NADH和基于NADP + 的还原测量NADP-ME活性。
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| RNA-Seq Library Generation from Rare Human Cells Isolated by FACS
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Author:
Date:
2013-06-20
[Abstract] High throughput RNA Sequencing has revolutionized transcriptome analyses. However, most available protocols require micrograms of RNA rendering this technique not feasible for analyzing small numbers of cells, including precious rare cell types isolated from human tissues or organs. Here, we used an RNA Amplification System and describe a method for preparing RNA sense-strand cDNA libraries compatible with an Illumina sequencing platform starting from limited numbers of human fetal germ cells as well as human embryonic stem cells (hESCs) isolated using Fluorescence Activated Cell Sorting (FACS). With this protocol we generated seven RNA-Seq libraries starting from 4,000 germ cells sorted from fetal ovaries (n = 2) and fetal testes (n = 2) at 16-16.5 weeks of development and 4,000 sorted ...
[摘要] 高通量RNA测序革新了转录组分析。 然而，大多数可用的协议需要微克RNA，使得这种技术不可能用于分析少量的细胞，包括从人体组织或器官分离的珍贵的稀有细胞类型。 在这里，我们使用RNA扩增系统，并描述了一种制备RNA有义链cDNA文库的方法，其与Illumina测序平台相容，从有限数目的人胎儿生殖细胞以及使用荧光活化细胞分离的人胚胎干细胞（hESC） 排序（FACS）。 使用这个协议，我们生成七个RNA Seq库开始从4,000生殖细胞从胎儿卵巢（n = 2）和胎儿睾丸（n = 2）在16-16.5周的发展和4,000分选hESCs（n = 3）排序。 我们预测多重文库也可以通过用多重兼容的3'衔接子和索引的PCR引物替换这里使用的单重3'衔接子来产生。
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