该方法基于在靶细胞类型中选择性表达血凝素 A (HA) 标记的核糖体蛋白，然后使用针对 HA 的抗体从靶细胞中纯化多核糖体和相关 mRNA。 最初的方法利用了带有 Rpl22 修饰等位基因 (Rpl22-HA) 的小鼠品系 (RiboTag)，该等位基因是由 Cre 重组酶的作用诱导的。

Ribosome tagging has become a very useful in vivo approach for analyzing gene expression and mRNA translation in specific cell types that are difficult and time consuming to isolate by conventional methods.

2021年3月19日 · Cell isolation may alter gene expression and is disruptive to mature neurons with extensive processes. This protocol describes cell-type-specific expression of tagged ribosome and the use of ribosome tagging followed by RNA-seq to identify translatome of low number and sparse cells in mouse brains without disruptive cell isolation.

Basic Introduction: The Ribogreen assay is able to quantify the total RNA content of your sample. Our yield is usually under 5 ng/μl for the IP samples and can be between 20 and 100 for the input sample. Considering this, remember to dilute your input sample in RNase-free water to allow it to fall solidly within your standard curve.

RiboTag IP was performed according to Sanz et al. 2019, adapted for intestinal tissue as follows: Small bowel whole intestinal tissue was placed in ice-cold Krebs-Henseleit buffer (Table 2 ), and muscle layer mechanically separated from the mucosal layer.

RiboTag co-transfection represents a convenient and powerful tool to isolate RNA from a specific subset of cultured cells with a variety of applications for experiments in vitro. Keywords: Cell-type specific gene expression, translational profiling, RiboTag, …

2015年6月1日 · Here we describe our adaptation of a ribosomal capture strategy that was designed to be used in transgenic mice expressing tagged ribosomal subunits (RiboTag) in specific cell types, thereby allowing measurement of translating RNAs from desired cell types within complex tissues.

IP of the tagged ribosomes from whole tissue extracts with anti-HA antibody-coupled magnetic beads enables the pull-down of cell type-specific ribosome-attached mRNA, i.e. the translatome. Here we report the application of the RiboTag approach to the study of microglia.

2019年6月1日 · To specifically isolate astrocyte mRNA in vivo from complex tissues, we used the ribotag method to genetically tag ribosomes in astrocytes, restricting analysis to astrocytes and enabling ...

In this protocol, we describe a ribosome-tagging approach (RiboTag) which our lab developed (Sanz et al., 2009) and discuss the application of this approach to the analysis of...