单细胞RNA测序,简称scRNA-seq,是一种让科学家能够研究混合群体中单个细胞中基因表达的技术, 而混合群体是所有细胞在人体组织中的存在的方式。
做为单细胞测序技术大家族的一部分,scRNA-seq涉及捕获单个细胞的RNA,并在多次分子转化反应后,对其进行测序。由于RNA是从DNA基因到蛋白质的中间步骤,它提供了一个关于特定细胞中哪些基因是活跃的,哪些不是活跃的描述。
因为scRNA-seq捕捉了细胞基因组中所有基因的活性,一次捕获数千个基因,它已经成为定义细胞状态和表型的金标准。这类数据可以揭示细胞群体中罕见的细胞类型,甚至是以前从未见过的类型。
但scRNA-seq不仅仅是一个用于基础细胞生物学的工具;由于它能够识别组织中哪些细胞正在积极分裂,或者哪些细胞对特定药物、或治疗有反应,因此在医学和药理学研究中被广泛采用。
这些单细胞方法改变了我们在系统间解析细胞特性的能力。
但是,研究人员碰到的问题是,scRNA seq目前是针对大单元输入定制的。
zhè这xiē些dān单xì细bāo胞fāng方fǎ法gǎi改biàn变le了wǒ我men们zài在xì系tǒng统jiān间jiě解xī析xì细bāo胞tè特xìng性de的néng能lì力。wèn问tí题shì是,tā它men们mù目qián前shì是zhēn针duì对dà大dān单yuán元shū输rù入dìng定zhì制de的。
This isn't a trivial problem, as scRNA-seq methods require over a thousand cells for a useful measurement. Dr Johannes Bues, a researcher in Deplancke's group, adds: "This renders them inefficient and costly when processing small, individual samples such as small tissues or patient biopsies, which tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs."
这不是一个小问题,因为scRNA-seq方法需要1000多个细胞才能进行有用的测量。这使得它们在处理小型个体样本(如小组织或患者活检)时效率低下,成本高昂,这些样本往往需要通过加载大量样本来解决,从而产生混乱的镶嵌细胞群读数。
zhè这bù不shì是yī一gè个xiǎo小wèn问tí题,yīn因wèi为scRNA-seqfāng方fǎ法xū需yào要1000duō多gè个xì细bāo胞cái才néng能jìn进xíng行yǒu有yòng用de的cè测liáng量。dé德pǔ普lǎng朗kè克yán研jiū究xiǎo小zǔ组de的yán研jiū究yuán员yuē约hàn翰nèi内sī斯·bù布sī斯bó博shì士bǔ补chōng充shuō说:“zhè这shǐ使de得tā它men们zài在chǔ处lǐ理xiǎo小xíng型gè个tǐ体yàng样běn本(rú如xiǎo小zǔ组zhī织huò或huàn患zhě者huó活jiǎn检)shí时xiào效lǜ率dī低xià下qiě且chéng成běn本gāo高áng昂,zhè这xiē些yàng样běn本wǎng往wǎng往tōng通guò过jiā加zài载dà大liàng量yàng样běn本lái来jiě解jué决,cóng从ér而chǎn产shēng生hùn混luàn乱de的xiāng镶qiàn嵌xì细bāo胞qún群dú读shù数。”
The DisCo solution
The DisCo solution解决方案
dí迪sī斯kē科jiě解jué决fāng方àn案
Bues, with Marjan Biočanin and Joern Pezoldt, also in Deplancke's group, have now developed a new method that allows scRNA-seq to efficiently process samples with fewer cells. Published in Nature Methods, the method is dubbed "DisCo" for "deterministic, mRNA-capture bead and cell co-encapsulation dropleting system".
瑞士联邦理工学院EPFL的研究人员Bues与Marjan Biočanin和Joern Pezoldt等人组成的团队现在已经开发出一种新方法,让scRNA seq以更少的细胞有效地处理样本。该方法发表在《自然方法》杂志上,被称为DisCo,deterministic, mRNA-capture bead and cell co-encapsulation dropleting system,一种液滴系统。

Buesyǔ与MarjanBiočaninhé和JoernPezoldt(yě也zài在dé德pǔ普lǎng朗kè克de的tuán团duì队zhōng中)xiàn现zài在yǐ已jīng经kāi开fā发chū出yī一zhǒng种xīn新fāng方fǎ法,yǔn允xǔ许scRNAseqyǐ以gèng更shǎo少de的xì细bāo胞yǒu有xiào效de地chǔ处lǐ理yàng样běn本。gāi该fāng方fǎ法fā发biǎo表zài在《zì自rán然fāng方fǎ法》zá杂zhì志shàng上,bèi被chēng称wéi为“DisCo”,yòng用yú于“què确dìng定xìng性、mRNAbǔ捕huò获zhū珠hé和xì细bāo胞gòng共bāo包mái埋yè液dī滴xì系tǒng统”。
Unlike usual single-cell methods that rely on passive cell capture, DisCo uses machine-vision to actively detect cells and capture them in droplets of oil and beads. This approach allows for continuous operation, and also renders scaling and serial processing of cell samples highly cost efficient.
与通常依靠被动细胞捕获的单细胞方法不同,DisCo利用机器视觉主动检测细胞,并将其捕获在油滴和珠子中。这种方法允许连续操作,并且使细胞样本的缩放和串行处理具有很高的成本效益。
yǔ与tōng通cháng常yī依kào靠bèi被dòng动xì细bāo胞bǔ捕huò获de的dān单xì细bāo胞fāng方fǎ法bù不tóng同,dí迪sī斯kē科lì利yòng用jī机qì器shì视jué觉zhǔ主dòng动jiǎn检cè测xì细bāo胞,bìng并jiāng将qí其bǔ捕huò获zài在yóu油dī滴hé和zhū珠zi子zhōng中。zhè这zhǒng种fāng方fǎ法yǔn允xǔ许lián连xù续cāo操zuò作,bìng并qiě且shǐ使xì细bāo胞yàng样běn本de的suō缩fàng放hé和chuàn串xíng行chǔ处lǐ理jù具yǒu有hěn很gāo高de的chéng成běn本xiào效yì益。
As shown in the study, DisCo features precise particle and cell positioning, and controls droplet sorting through combined machine-vision and multilayer microfluidics. All this allows for continuous processing of low-input single cell suspensions at high capture efficiency (over 70%) at speeds that can reach 350 cells per hour.
如研究所示,DisCo具有精确的粒子和细胞定位功能,并通过组合机器视觉和多层微流体控制液滴分选。所有这些都允许以高捕获效率(超过70%)连续处理低输入单细胞悬浮液,速度可达每小时350个细胞。
rú如yán研jiū究suǒ所shì示,DisCojù具yǒu有jīng精què确de的lì粒zǐ子hé和xì细bāo胞dìng定wèi位gōng功néng能,bìng并tōng通guò过zǔ组hé合jī机qì器shì视jué觉hé和duō多céng层wēi微liú流tǐ体kòng控zhì制yè液dī滴fēn分xuǎn选。suǒ所yǒu有zhè这xiē些dōu都yǔn允xǔ许yǐ以gāo高bǔ捕huò获xiào效lǜ率(chāo超guò过70%)lián连xù续chǔ处lǐ理dī低shū输rù入dān单xì细bāo胞xuán悬fú浮yè液,sù速dù度kě可dá达měi每xiǎo小shí时350gè个xì细bāo胞。
To further showcase DisCo's unique capabilities, the researchers tested it on the small chemosensory organs of the Drosophila fruit fly, as well as on individual intestinal crypts and organoids. The latter are tiny tissues grown in culture dishes closely resembling actual organs – a field that EPFL has been spearheading for years.
为了进一步展示DisCo的独特功能,研究人员在果蝇的小型化学感觉器官以及单个肠隐窝和类器官上进行了测试。后者是在培养皿中生长的微小组织,与实际器官非常相似,EPFL多年来一直在这一领域处于领先地位。
wèi为le了jìn进yī一bù步zhǎn展shì示dí迪sī斯kē科de的dú独tè特gōng功néng能,yán研jiū究rén人yuán员zài在guǒ果yíng蝇de的xiǎo小xíng型huà化xué学gǎn感jué觉qì器guān官yǐ以jí及dān单gè个cháng肠yǐn隐wō窝hé和lèi类qì器guān官shàng上jìn进xíng行le了cè测shì试。hòu后zhě者shì是zài在péi培yǎng养mǐn皿zhōng中shēng生zhǎng长de的wēi微xiǎo小zǔ组zhī织,yǔ与shí实jì际qì器guān官fēi非cháng常xiāng相sì似——EPFLduō多nián年lái来yī一zhí直zài在zhè这yī一lǐng领yù域chǔ处yú于lǐng领xiān先dì地wèi位。
The researchers used DisCo to analyze individual intestinal organoids at different developmental stages. The approach painted a fascinating picture of heterogeneity in the organoids, detecting various distinct organoid subtypes of which some had never been identified before.
研究人员使用DisCo分析了不同发育阶段的个体肠道类器官。该方法描绘了类器官异质性的画面,检测出各种不同的类器官亚型,其中一些亚型以前从未被确定。
yán研jiū究rén人yuán员shǐ使yòng用DisCofēn分xī析le了bù不tóng同fā发yù育jiē阶duàn段de的gè个tǐ体cháng肠dào道lèi类qì器guān官。gāi该fāng方fǎ法miáo描huì绘le了lèi类qì器guān官yì异zhì质xìng性de的mí迷rén人huà画miàn面,jiǎn检cè测chū出gè各zhǒng种bù不tóng同de的lèi类qì器guān官yà亚xíng型,qí其zhōng中yī一xiē些yà亚xíng型yǐ以qián前cóng从wèi未bèi被què确dìng定。
"Our work demonstrates the unique ability of DisCo to provide high-resolution snapshots of cellular heterogeneity in small, individual tissues," says Deplancke.
该研究工作证明了DisCo独特的能力,能够提供小而个体组织中细胞异质性的高分辨率快照。
参考文献
Deterministic scRNA-seq captures variation in intestinal crypt and organoid composition
相关阅读
单细胞RNA测序揭示药物诱导的干细胞背叛