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Jun 2020
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Mechanical Fractionation of Cultured Neuronal Cells into Cell Body and Neurite Fractions
培养神经元细胞成细胞体和神经突部分的机械分离   

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Abstract

Many cells contain spatially defined subcellular regions that perform specialized tasks enabled by localized proteins. The subcellular distribution of these localized proteins is often facilitated by the subcellular localization of the RNA molecules that encode them. A key question in the study of this process of RNA localization is the characterization of the transcripts present at a given subcellular location. Historically, experiments aimed at answering this question have centered upon microscopy-based techniques that target one or a few transcripts at a time. However, more recently, the advent of high-throughput RNA sequencing has allowed the transcriptome-wide profiling of the RNA content of subcellular fractions. Here, we present a protocol for the isolation of cell body and neurite fractions from neuronal cells using mechanical fractionation and characterization of their RNA content.


Graphic abstract:



Fractionation of neuronal cells and analysis of subcellular RNA contents


Keywords: RNA localization (RNA定位), RNA transport (RNA转运), Post-transcriptional regulation (转录水平后的调控), RNA trafficking (RNA运输), Subcellular transcriptomics (亚细胞转录组学)

Background

In eukaryotic cells, proteins are asymmetrically distributed to define spatially specialized regions. In cells that have complex morphologies and/or large sizes,like neurons, this asymmetry is often more extreme. These extended morphologies require a regulated and efficient sorting process to ensure that proteins are correctly localized. For many proteins, this process is facilitated through the transport of RNA molecules to the site of protein function (Engel et al., 2020). On-site translation of these RNAs produces a protein that is immediately correctly localized. In neurons, this is a widespread process as up to 1000 different RNA species are enriched in the projections of these cells relative to their cell bodies (Cajigas et al., 2012; Taliaferro et al., 2016).


RNA localization is widely used as a gene expression regulatory strategy and contributes to a diverse set of biological processes, including mating-type switching in yeast (Bertrand et al., 1998), developmental patterning in Drosophila (Ephrussi et al., 1991; Lécuyer et al., 2007), and nutrient response in intestinal epithelial cells (Moor et al., 2017). The misregulation of RNA localization is associated with a range of neurological diseases (Wang et al., 2016), including spinal muscular atrophy (Fallini et al., 2011), amyotrophic lateral sclerosis (Chu et al., 2019; Briese et al., 2020), and Fragile X Syndrome (Dictenberg et al., 2008; Goering et al., 2020).


Despite the increasing recognition of the role of RNA localization in promoting a range of cellular functions, several questions remain unanswered. Among them is perhaps one of the simplest questions that exists regarding RNA localization: what RNAs exist at a given subcellular location and what are their relative abundances? To answer this question, at least in the context of neuronal cells, a variety of techniques have been developed and applied, including laser capture microdissection (Zivraj et al., 2010), compartmentalized culture chambers (Gumy et al., 2011), growth on microfluidic devices (Nijssen et al., 2018), and microdissection of rodent brains (Cajigas et al., 2012).


In this protocol, we describe a similar approach to separate neuronal cells into neurite and cell body fractions. This technique relies on microporous culture membranes. Cells are cultured on top of these membranes, which have pores (usually 1-3 μm in diameter) large enough to allow neurites to pass through them to the underside but small enough to keep the cell bodies on the top of the membrane. After growth on the membranes, the cells can be mechanically fractionated by scraping the top of the membrane with a cell scraper and removing the dislodged cell bodies. Neurites remain attached to the underside of the membrane and can be lysed for RNA extraction. Following RNA isolation, the RNA content of the fractions can be analyzed by reverse transcription-quantitative PCR (RT-qPCR) or by high-throughput RNA sequencing.


Although the focus of this manuscript is the fractionation of neuronal cells, this method can also be applied to cellular protrusions from a variety of cell types, including fibroblasts (Mili et al., 2008) and migrating cancer cells (Mardakheh et al., 2015; Dermit et al., 2020). In the context of neuronal cells, this procedure has been successfully used with neuronal cell lines (Taliaferro et al., 2016; Goering et al., 2020), primary mouse cortical neurons (Taliaferro et al., 2016), and iPS-derived neurons (Goering et al., 2020; Hudish et al., 2020). It is likely compatible with most neuronal cell types.

Materials and Reagents

  1. Deep well 6-well cell culture plates (Corning, catalog number: 353502, store at room temperature)

  2. Microporous transwell membranes (transwell cell culture inserts), 1 μm pore diameter (Corning, catalog number: 353102, store at room temperature)

  3. Matrigel (VWR, catalog number: 47743-706, store at -20°C)

  4. RNase-free pipetting equipment (e.g., aerosol-resistant filter tips)

  5. Cell lifter/scraper (Fisher, catalog number: 07-200-364, store at room temperature)

  6. Quick RNA Microprep kit (Zymo, catalog number: R1051, store at room temperature)

  7. Cell culture media (dependent on the needs of the specific cells being grown)

  8. Mouse anti-beta actin antibody (Sigma, catalog number: A5441)

  9. Mouse anti-histone H3 antibody (Abcam, catalog number: 10799)

  10. iScript reverse transcription supermix (BioRad, catalog number: 1708841)

  11. Taqman probes and master mix (ThermoFisher, catalog number: 4444556)

  12. MOPS running buffer (Invitrogen, catalog number: NP0001)

  13. Protein sample buffer (Invitrogen, catalog number: NP0008, store at room temperature)

Equipment

  1. Cell culture tabletop centrifuge (for example, Eppendorf, model: 5702R)

  2. Benchtop microcentrifuge (for example, Eppendorf, model: 5424R)

  3. SDS page and western blotting materials

  4. Thermocycler

  5. qPCR-enabled thermocycler

Procedure

  1. Prepare transwell membranes

    1. Dilute Matrigel to 0.2% in cell culture media (see Note 1).

    2. Place the transwell membranes upside down in a 15 cm cell culture plate. Add 1 ml of diluted matrigel on the top, coating the bottom (underside) of each transwell membrane.

    3. Incubate at 37°C for 1 h.


  2. Prepare cells

    1. In the meantime, wash cells with 1× PBS and trypsinize them.

    2. Pellet by centrifuging at 500 × g for 5 min.

    3. Resuspend in cell culture media to a concentration of 500,000 cells per ml. Two milliliters of this cell suspension is needed for each transwell membrane (see Note 2).


  3. Plate cells

    1. Remove Matrigel solution from the transwell membranes.

    2. In each well of a deep well 6-well plate, place 4 ml of cell culture media.

    3. Put one transwell filter in each well.

    4. Place 2 ml of cell solution (Step B3) onto each filter.

    5. If a media change following plating is necessary (e.g., a change into differentiation-inducing media), allow cells to attach for 1 h, then replace the media above and below the membrane. Change pipettes in between dealing with the solutions above and below the membrane to avoid introducing cells into the lower chamber.

    6. Allow cells to incubate at 37°C for 48 h.


  4. Fractionate cells

    Note: For a description of this procedure, see Video 1.


    Video 1. Mechanical fractionation of neuronal cells for subcellular RNA analysis. This video details the procedures for mechanical fractionation of neuronal cells using microporous membranes and highlights key points of the protocol.


    1. Gently remove media above and below the membrane by aspiration.

    2. Replace the media with PBS, using 2 ml below the membrane and 1 ml above the membrane.

    3. Remove the cell bodies on the top of each membrane.

      1. Gently but thoroughly scrape the top of each membrane with a cell lifter, making sure to get the edges of the membrane where it joins the plastic housing (Figure 1A) (see Note 3).



        Figure 1. Mechanical fractionation of cells. A. The top of the membrane is scraped to remove cell bodies. B. The membrane is then removed from the plastic housing using a razor blade. C. The cut membrane is placed in a dish of lysis buffer using tweezers.


      2. Transfer 700 μl of the cell body suspension into a 15 ml conical tube on ice.

      3. Tilting the membrane, scrape any remaining cell bodies into the remaining 300 μl of PBS.

      4. Thoroughly transfer the PBS into the 15 ml conical tube.

      5. Place the membrane upside down on a clean surface (for example, the lid of the 6-well plate).

      6. Repeat Steps D3a through D3e for the remaining membranes.

    4. After scraping and removing the cell bodies from all six membranes, place 550 μl of RNA lysis buffer from the Zymo RNA Microprep kit into a 6 cm dish.

    5. Remove each membrane from its plastic housing.

      1. Using a fresh razor blade, cut each membrane to remove it from the housing, leaving approximately 3 mm around the edge (Figure 1B) (see Note 4).

      2. Using tweezers, carefully put the released membrane into the RNA lysis buffer with the neurite-containing side facing down (Figure 1C).

      3. Repeat for all six membranes, placing all in the same 6 cm dish.

    6. Incubate the dish at room temperature with rocking for 15 min to lyse neurites.

    7. In the meantime, centrifuge cell bodies at 2,000 × g at 4°C for 7 min. Resuspend in 600 μl of PBS (i.e., 100 μl per membrane).

    8. Reserve samples for western blotting.

      1. Take 10 μl of the 600 μl cell body suspension. Add 90 μl of Protein Sample Buffer.

      2. Take 50 μl of the 550 μl neurite lysate. Add to 50 μl of Protein Sample Buffer. Guanidine in the RNA lysis buffer will precipitate from solution upon addition to Protein Sample Buffer, but that is expected.

      3. Store these samples at -20°C until analysis of fractionation efficiency by western blotting.

    9. Isolate RNA

      1. Take 100 μl of cell body suspension and isolate RNA according to the instructions of the Zymo Quick RNA Microprep kit, beginning with the addition of 350 μl of RNA lysis buffer.

      2. Take the remaining 500 μl of neurite lysate and isolate RNA according to the kit’s instructions, beginning with the addition of 500 μl of 95-100% ethanol.

    10. Elute RNA

      Elute RNA in 15 μl RNase free water (see Note 5).


  5. Analysis of fractionation efficiency using western blotting (see Note 6)

    1. Heat the samples that were reserved for western blotting (Step D8) for 5 min at 98°C.

    2. Load 5 μl of cell body sample per lane.

    3. Load 15 μl of neurite sample per lane while it is still hot. This helps any precipitated guanidine to dissolve and facilitates loading.

    4. Run a SDS-PAGE gel and transfer proteins to a nitrocellulose or PVDF membrane.

    5. Blot with primary antibodies.

      1. Use the β-actin antibody at 1:5,000 dilution.

      2. Use the histone H3 antibody at 1:10,000 dilution.

    6. Probe with an appropriate mouse secondary antibody.

    7. Image blot. See Figure 2 for a blot depicting an efficient fractionation.



      Figure 2. Assessment of fractionation efficiency by western blotting. Cell body samples are indicated by CB, and neurite samples are indicated by N.


  6. Analysis of fractionation efficiency using RT-qPCR (see Note 7)

    1. Reverse transcribe 100 ng of RNA from each sample (see Note 8)

      1. Combine 2 μl of 5× iScript RT master mix, RNA, and water into a 10 μl reaction.

      2. Incubate in a thermocycler for 5 min at 25°C, 20 min at 46°C, and 1 min at 95°C.

      3. Dilute reaction with water to a 20 μl final volume (see Note 9).

    2. Perform qPCR

      Using a qPCR master mix of your choice, perform qPCR to quantify the desired marker RNA molecules in each fraction. Use 2 μl of cDNA from Step F1c per reaction. It is usually best to quantify the ratio of two RNA molecules in each sample, with one of the two known to be neurite enriched. The accuracy of the ratio in each sample can be improved using TaqMan qPCR probes to allow simultaneous quantification of the two species in the same reaction.

    3. Assess qPCR results

      From the qPCR results, calculate the relative abundance of the neurite-enriched and control RNAs in the cell body and neurite fractions. If the fractionation was successful, the ratio of the neurite-enriched RNA to control RNA will be higher in the neurite fraction than in the cell body fraction. See Figure 3 for qPCR results from a successful fractionation. The increased variation in neurite quantification relative to cell body quantification is typical and likely reflects the technical variation inherent to differences in fractionation efficiency between replicates.



      Figure 3. qPCR results from a successful fractionation. Ranbp1 RNA is known from previous experiments to be enriched in neurites, while Tsc1 is known from previous experiments to be enriched in cell bodies. The relative amounts of these RNA species were quantified in cell body and neurite samples using Taqman qPCR.


  7. Construction of high-throughput sequencing libraries

    Use the purified RNA to make high-throughput RNA sequencing libraries. Several commercial kits are available for this purpose, although we have had consistently good results using the mRNA Hyperprep kit from KAPA (Kapa KK8580). Importantly, provide the same amount of input RNA (e.g., 100 ng) for all samples in the kit, even though significantly more RNA can be isolated from cell bodies than from neurites.

Data analysis

  1. Analysis of western blot results can be done by visual inspection. Generally, there should be very little to no signal from histone H3 in the neurite samples. Significant histone H3 signal in the neurite samples is indicative of a poor fractionation. The level of β-actin signal may or may not be similar between the cell body and neurite fractions. What is important to consider is the relative ratio of β-actin to histone H3 signal in the two fractions.

  2. Identification of RNAs that are differentially localized between the two fractions can be done using standard differential gene expression techniques with high-throughput sequencing data.

Notes

  1. We recommend thawing the Matrigel stock overnight in a 2°C to 8°C refrigerator. Additionally, dilute matrigel in cold cell culture media as it starts to form a gel at 10°C. As matrigel is very viscous, aspirate and dispense the stock slowly.

  2. This concentration of cells ensures that they are essentially confluent when plated on the membrane. This can be desirable because it can result in neurite outgrowth being forced down through the pores of the membrane rather than laterally across the surface. If this is not desirable, adjust the cell concentration accordingly.

  3. Using too much pressure when scraping can result in the membrane being torn away from the plastic housing. If this happens, discard the torn membrane and move to the next one.

  4. It is often difficult to completely remove cell bodies from the corner formed by the membrane and the plastic housing. For this reason, it is often best to avoid this area when cutting the membrane out of the housing. Do not worry about removing all of the membrane when cutting.

  5. When this procedure is performed with N2A or CAD mouse neuronal cell lines, expect 5-10 μg of RNA from the cell body sample and 500-1,000 ng of RNA from the neurite sample. This is the expected amount when all wells of a 6-well plate are combined.

  6. The fractionation efficiency can be assessed by probing the cell body and neurite fractions for specific proteins. β-Actin should be present in both fractions, whereas histone H3, being nuclear, should be restricted to the cell body fraction. The detection of significant histone H3 signal in the neurite fraction indicates poor fractionation efficiency. The high amount of salt in the neurite fractions may cause them to appear compressed during imaging or while running the gel. This is a purely cosmetic defect, and the ability to detect protein bands within these samples is not hindered.

  7. The efficiency of fractionation can also be assessed using RT-qPCR. This requires knowledge of RNA species enriched in each fraction. We have observed that mRNAs encoding ribosomal proteins are reproducibly neurite-enriched across several neuronal cell types and species. In this example, we use Ranbp1 and Tsc1 RNAs as markers, which we have previously observed to be neurite-enriched and cell body-enriched, respectively.

  8. It should be noted that qPCR requires ~100 ng RNA from both cell body and neurite fractions. Cells from two wells are enough to serve as one replicate for qPCR. Thus, a full 6-well plate can be split into three replicates for qPCR. We highly recommend performing a minus RT control to ensure that there is no genomic DNA contamination in the samples.

  9. The dilution factor for the RT reaction depends on the expression of the genes that need to be tested. For highly expressed genes, the RT reaction can be diluted to 50 μl final volume.

Acknowledgments

This work was funded by the National Institutes of Health (R35-GM133885) (JMT), the Boettcher Foundation (Webb-Waring Early Career Investigator Award AWD-182937), a Predoctoral Training Grant in Molecular Biology (NIH-T32-GM008730) (RG), and the RNA Bioscience Initiative at the University of Colorado Anschutz Medical Campus (RG and JMT). The protocol presented here was derived from previous work published by the authors (Taliaferro et al., 2016; Goering et al., 2020).

Competing interests

The authors declare no financial or non-financial competing interests.

Ethics

No animal or human subjects were used during this study.

References

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简介

[摘要]许多细胞包含在空间上定义的亚细胞区域,这些区域执行 由局部蛋白质实现的特殊任务。这些定位蛋白的亚细胞分布通常通过编码它们的RNA分子的亚细胞定位来促进。RNA定位过程研究中的一个关键问题是给定亚细胞位置上存在的转录本的特征。从历史上看,旨在回答这个问题的实验都集中在基于显微镜的技术上,该技术一次靶向一个或几个转录本。但是,最近,高通量RNA测序技术的出现允许对亚细胞级分的RNA内容进行转录组范围内的分析。在这里,我们使用机械分馏提出了一个协议用于细胞主体和从神经元细胞的神经突的级分的分离和 他们的RNA含量的表征。



图形摘要:





˚F神经元细胞的ractionation和亚细胞RNA内容分析

[背景技术在真核细胞中,蛋白质不对称分布以定义空间上专用的区域。在具有复杂的形态和/或大尺寸的细胞,像神经元,这种不对称通常更极端。这些扩展的形态要求有序的,有效的分选过程,以确保蛋白质正确定位。对于许多蛋白质,该过程通过将RNA分子转运到蛋白质功能位点而得以促进(E ngel等,202 0)。这些RNA的现场翻译产生立即被正确定位的蛋白质。在神经元中,这是一个普遍的过程,因为相对于它们的细胞体,这些细胞的投影中最多富集了1000种不同的RNA种类(Cajigas等,2012; Taliaferro等,2016)。

RNA定位被广泛用作基因表达的调节策略,并有助于一组不同的生物过程,包括交配-酵母型开关(贝特朗等人,发育模式中,1998年。)果蝇(埃弗吕西等人,1991; LECUYER等人(2007),以及肠道上皮细胞的养分响应(Moor等人,2017)。RNA定位的失调与一系列神经系统疾病(Wang等,2016)有关,包括脊髓性肌萎缩(Falini等,2011),肌萎缩性侧索硬化症(Chu等,2019; Briese等。 (2020)和脆弱X综合征(Dictenberg等,2008; Goering等,2020)。

尽管人们越来越认识到RNA本地化在促进一系列细胞功能中的作用,但仍有几个问题尚未解答。其中也许是最简单的问题存在一个小号方面荷兰国际集团RNA本地化:在给定的亚细胞定位存在哪些RNA和他们有什么相对丰度?为了回答这个问题,至少在神经元细胞的背景下,已经开发并应用了多种技术,包括激光捕获显微切割术(Zivraj等人,2010),分隔培养室(Gumy等人,2011),生长微流控设备(Nijssen et al 。,2018)和啮齿动物大脑的显微解剖(Cajigas et al 。,2012)。

在此协议中,我们描述了将神经元细胞分为神经突和细胞体部分的类似方法。该技术依赖于微孔培养膜。细胞在这些膜的顶部培养,其具有孔(通常是1-3微米直径)足够大,以允许突起通过它们传递到下侧,但足够小以保持细胞体在膜的顶部。上生长后的膜,可以将细胞机械地通过刮擦用细胞刮刀在膜的顶部,并除去脱落细胞体分馏。神经突仍然附着在膜的下面,可以裂解以提取RNA。以下RNA分离,所述级分的RNA含量能够通过反转录定量PCR(RT-qPCR的)或通过高被分析-可以通过RNA测序。

尽管此手稿的重点是神经元细胞的分离,但该方法也可以应用于多种细胞类型的细胞突起,包括成纤维细胞(Mili等人,2008)和迁移的癌细胞(Mardakheh等人,2015)。 ; Dermit等人,2020)。在神经元细胞的情况下,此程序已成功用于神经元细胞系(Taliaferro等人,2016 ;Goering等人,2020 ),原代小鼠皮层神经元(Taliaferro等人,2016)和iPS衍生神经元(Goering等,2020; Hudish等,2020)。它可能与大多数神经元细胞类型兼容。

关键字:RNA定位, RNA转运, 转录水平后的调控, RNA运输, 亚细胞转录组学



材料和试剂


深阱6 -孔细胞培养板(Corning,目录号:353502,保存于室温下)
微孔transwell膜(transwell细胞培养插入物),孔径为1μm (Corning,目录号:353102,在室温下保存)
基质胶(VWR,目录号:47743-706,储存在-20°C)
不含RNase的移液设备(例如,耐气溶胶的过滤嘴)
细胞提升器/刮板(Fisher,目录号:07-200-364,在室温下存储)
Quick RNA Microprep试剂盒(Zymo ,目录号:R1051,在室温下保存)
细胞培养基(取决于正在生长的特定细胞的需求)
小鼠抗β肌动蛋白抗体(西格玛,目录号:A5441)
小鼠抗组蛋白H3抗体(Abcam,目录号:10799)
iScript逆转录超混合(BioRad ,目录号:1708841)
Taqman探针和预混液(ThermoFisher ,目录号:4444556)
MOPS运行缓冲区(Invitrogen,目录号:NP0001)
蛋白质样品缓冲液(Invitrogen,目录号:NP0008,在室温下保存)


设备


细胞培养台式离心机(例如,Eppendorf ,型号:5702R)
台式微量离心机(例如,Eppendorf ,型号:5424R)
SDS页面和蛋白质印迹材料
热循环仪
支持qPCR的热循环仪


程序


准备transwell膜
稀基质胶以在细胞培养基中0.2%(小号EE注1)。
将transwell膜倒置在15 cm细胞培养板中。在t op上添加1 ml稀释的基质胶,包被每个transwell膜的底部(底侧)。
在37°C下孵育1小时。


准备细胞
同时,用1 × PBS洗涤细胞并用胰蛋白酶消化。
通过以500 × g离心5分钟来沉淀。 
重悬于细胞培养基中至每毫升500,000个细胞的浓度。两毫升该细胞悬浮液是需要针对每个跨孔膜(参见Ñ OTE 2)。


平板细胞
取下基底膜解决跨孔膜。
在每个孔的深阱6 -孔板中,代替4- ml的细胞培养介质。
在每个孔中放置一个Transwell过滤器。
的Plac e 2的毫升的细胞溶液(小号TEP乙3)到每个滤波器。
如果需要在铺板后更换培养基(例如,更换为诱导分化的培养基),请让细胞贴壁1小时,然后更换膜上方和下方的培养基。在处理之间变化移液器的溶液小号的上方和下方的膜,以避免introduc荷兰国际集团细胞进入下部腔室。
让细胞在37°C下孵育48小时。


分离细胞
Ñ OTE:˚F或此过程的说明,请参见视频1 。






视频1.神经元细胞的机械分离,用于亚细胞RNA分析。该视频详细说明了使用微孔膜和神经细胞的机械分离的过程凸显关键点的协议。


ģ ently ř EMOVE介质上方和下方的MEMB通过抽吸RANE。
用PBS代替培养基,在膜下2 ml,在膜上1 ml。
ř EMOVE上的顶部的细胞体的每个膜。
轻轻但彻底刮每个膜的顶部与细胞升降器,确保得到,它加入塑料壳体(图的膜的边缘1 A)(见注3)。






图1 。机械分馏细胞。一。刮擦膜的顶部以除去细胞体。乙。然后使用剃须刀片将薄膜从塑料外壳上取下。Ç 。使用镊子将切下的膜置于裂解缓冲液盘中。


转移700微升细胞悬浮液体的在冰上到15ml锥形管中。
倾斜膜,刮去任何剩余的细胞体到剩余的300微升PBS中。
将PBS彻底转移到15 ml锥形管中。
放置在一个干净的表面上膜颠倒(例如,6个的盖-孔板)。
重复小号TEP小号d 3的贯通D3E对于其余膜。
刮并从所有除去细胞体后6层膜,地点550微升从RNA裂解缓冲液的ZYMO RNA Microprep试剂盒为6cm皿。
从其塑料外壳中取出每个膜。
使用新鲜的剃须刀切割每个膜,将其从外壳上取下,在边缘周围留出约3 mm的距离(图1 B)(请参见注4)。
使用镊子,小心地将释放的膜放入含RNA的一面朝下的RNA裂解缓冲液中(图1 C)。
对所有六个膜重复上述操作,将所有膜放置在相同的6厘米培养皿中。
孵育所述在室温下培养皿摇动15分钟,以裂解神经突。
同时,将细胞体在4°C下于2,000 × g离心7分钟。重悬600微升的PBS(即,100微升每膜)。
保留样品进行Western blotting 。
采取10微升的该600微升细胞悬浮液体。加入90微升蛋白样品缓冲液。
采取50微升的该550微升神经突裂解物。添加到50μl蛋白质样品缓冲液中。加入蛋白质样品缓冲液后,RNA裂解缓冲液中的胍将从溶液中沉淀出来,但这是可以预料的。
将这些样品保存在-20°C下,直到通过蛋白质印迹分析分离效率为止。
分离RNA
取100微升细胞悬浮液体和分离RNA的根据的说明ZYMO快速RNA Microprep试剂盒,通过添加350的开始微升RNA裂解缓冲液中。
取剩余的500微升神经突裂解物和分离物的RNA根据试剂盒的说明书,添加500的开始微升的95-100%的乙醇。
洗脱RNA
在15μl无RNase的水中洗脱RNA (请参见注释5)。


使用蛋白质印迹分析分馏效率(参见注释6)
在98°C加热保留用于蛋白质印迹的样品(S tep D8)5分钟。
负载5微升每泳道细胞体的样品。
负载15微升每泳道神经突样品而它仍然是热的。这有助于任何沉淀的胍溶解并促进负载。
运行一个SDS-PAGE凝胶并转移蛋白到一个硝酸纤维素或PVDF膜。
用一抗进行印迹。
使用1的β-actin抗体:5 ,000稀释。
以1 : 10,000稀释度使用组蛋白H3抗体。
与探针的适当小鼠二级抗体。
图像印迹。参见图2描绘了有效分馏的印迹。






图2 。通过蛋白质印迹评估分级效率。CB表示细胞体样本,N表示神经突样本。


使用RT-qPCR分析分级效率(请参见注释7)
从每个样品中反转录100 ng RNA(请参见注释8)
结合2微升5 ×的iScript RT主混合物,RNA,和水加入到一个10微升的反应。
孵育在25℃,在46分钟20℃,5分钟热循环和在95℃下1分钟。
稀反应用水至一个20微升最终体积(见注9)。
进行定量PCR
使用您选择的qPCR预混液,执行qPCR以定量每个馏分中所需的标记RNA分子。使用2个微升从cDNA的小号TEP F1C每反应。通常最好量化每个样品中两个RNA分子的比例,已知其中两个是神经突富集的。使用TaqMan qPCR探针可以提高每个样品中比率的准确性,从而可以在同一反应中同时定量两种物质。


评估qPCR结果
从qPCR结果中,计算细胞体和神经突部分中富含神经突的RNA和对照RNA的相对丰度。如果分级成功,则在神经突部分中富含神经突的RNA与对照RNA的比率将比在细胞体部分中高。成功分馏后的qPCR结果见图3 。相对于细胞体定量,神经突定量变化的增加是典型现象,并且可能反映了重复样本之间分离效率差异所固有的技术变化。






图3 。qPCR来自成功的分级分离。从先前的实验中已知Ranbp1 RNA富含神经突,而从先前的实验中已知Tsc1富含细胞体。使用Taqman qPCR在细胞体和神经突样品中定量这些RNA种类的相对量。


高通量测序文库的构建
使用纯化RNA,使高-通量RNA测序文库。尽管我们一直使用KAPA的mRNA Hyperprep试剂盒(Kapa KK8580)获得了良好的结果,但仍有几种商用试剂盒可用于此目的。重要的是,提供输入RNA(相同量的例如,对于在试剂盒中的所有样本100纳克),即使显著多种RNA可以从比从神经突的细胞体中分离出来。


数据分析


蛋白质印迹结果的分析可以通过目测进行。通常,在神经突样本中,来自组蛋白H3的信号应该很少甚至没有。小号神经突样品中ignificant组蛋白H3信号指示差分馏的。细胞体和神经突部分之间的β-肌动蛋白信号水平可能相似,也可能不相似。重要的是要考虑这两个部分中β-肌动蛋白与组蛋白H3信号的相对比率。
我被两个组分之间差异局部RNA的dentification可以使用标准的差异基因表达技术来完成用高通量测序数据。


笔记


我们建议将Matrigel原料在2°C至8°C的冰箱中融化过夜。此外,在冷细胞培养基中稀释基质胶,因为基质胶会在10°C下开始形成凝胶。由于Matrigel非常粘稠,因此请缓慢吸出并分配股票。
细胞的这种浓度确保了当铺在膜上时它们基本上是汇合的。这可能是理想的,因为它可能导致神经突的生长被迫向下穿过膜的孔,而不是横向穿过整个表面。如果不希望如此,请相应地调整细胞浓度。
刮擦时使用太大的压力会导致薄膜从塑料外壳上撕下。如果发生这种情况,请丢弃破损的膜,然后移至下一个。
通常难以从膜和塑料外壳形成的角部完全去除细胞体。因此,从外壳上切下薄膜时通常最好避开该区域。不要担心删除所有的膜时切割。
当与N2A或CAD小鼠神经元细胞系中进行该过程,期望5-10微克的RNA从细胞体样品和500-1 ,000从神经突样品RNA的纳克。这是当一个6的所有孔中的预期量-孔板被组合。
可以通过探测特定蛋白质的细胞体和神经突部分来评估分离效率。β-甲一个已将应存在于两个级分,而组蛋白H3,作为核,应限制到电池单元主体部分。在神经突部分中检测到显着的组蛋白H3信号表明差的分离效率。神经突部分中大量的盐可能导致它们在成像过程中或凝胶电泳时看起来受压。这是纯粹的美容缺陷,检测这些样品中蛋白质条带的能力没有受到阻碍。
分馏的效率也可以使用RT-qPCR进行评估。这需要了解每个部分都富集的RNA种类。我们已经观察到,编码核糖体蛋白的mRNA在多个神经元细胞类型和物种中可再生地富含神经突。在此示例中,我们使用Ranbp1和Tsc1 RNA作为标记,我们之前已经观察到它们分别被神经突富集和细胞体富集。
应当指出,qPCR需要从细胞体和神经突部分中提取约100 ng RNA。来自两个孔的细胞足以用作qPCR的一个复制品。因此,可以将完整的6孔板分成三份重复进行qPCR。我们强烈建议执行负RT控制,以确保样品中没有基因组DNA污染。
RT反应的稀释因子取决于需要测试的基因的表达。对于高度表达的基因,RT反应可以稀释至50μl最终体积。


致谢


这项工作由美国国立卫生研究院(R35-GM133885),Boettcher基金会(韦伯战争早期职业研究者奖AWD-182937),分子生物学博士前培训补助金(NIH-T32-GM008730)资助( RG),以及位于科罗拉多大学安舒兹分校(RG和JMT)的RNA生物科学计划。此处介绍的协议源自作者先前的工作(Taliaferro等人,2016;Goering等人,2020)。


利益争夺


作者声明没有任何金融或非金融竞争利益。


伦理


在这项研究中没有使用动物或人类受试者。


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Copyright Arora et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Arora, A., Goering, R., Lo, H. G. and Taliaferro, J. M. (2021). Mechanical Fractionation of Cultured Neuronal Cells into Cell Body and Neurite Fractions. Bio-protocol 11(11): e4048. DOI: 10.21769/BioProtoc.4048.
  2. Goering, R., Hudish, L. I., Guzman, B. B., Raj, N., Bassell, G. J., Russ, H. A., Dominguez, D. and Taliaferro, J. M. (2020). FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex RNA sequences. Elife 9: e52621.
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