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- 提供商：
康测科技
- 服务名称：
绝对定量转录组测序
绝对定量转录组测序（UMI RNA-seq）
RNA&测序
- 生物体细胞内存在着多种RNA，以真核生物为例，除了编码蛋白质的mRNA之外，还存在大量非编码的RNA。根据其功能又可以分为house keeping ncRNA（rRNA和tRNA）和Regulatory ncRNA。Regulatory RNA近年来被发现行使多种复杂的调控功能，是当前以及未来生物学研究的重点。
- 根据这些RNA的结构及长度，又可以分为长链非编码RNA（lncRNA）、小RNA（siRNA、miRNA、piRNA、snoRNA和snRNA）以及环状RNA（circRNA）。
这些RNA各自具有不同的生物学功能，为了研究这些RNA的功能，目前发展出了多种测序技术对其表达进行检测。
| 测序类型 | mRNA-seq | lncRNA-seq | sRNA-seq | circRNA-seq |
| 技术方案 | polyA捕获 | rRNA剔除 | 连接富集 | 去除线性RNA |
| 检测对象 | mRNA pre-mRNA(部分) lncRNA(部分) |
mRNA pre-mRNA lncRNA circRNA(部分） |
miRNA siRNA piRNA(部分) |
circular RNA |
技术痛点
测序结果的准确性受到文库扩增时PCR偏好性的极大影响：
- RNA相关测序技术在文库的构建过程中都需要使用PCR扩增，从而产生重复的分子片段；
- 在测序过程的PCR扩增中，也会产生重复的分子片段。
- 但是所有的文库片段并非以同等速率等量的扩增，扩增速率受到片段长度、GC含量、片段浓度等多方面的影响，容易扩增的片段极大的被富集，一些含量较低的片段或碱基偏好严重的片段甚至完全丢失，最终影响测序结果的准确性。
解决方案
KC-digital RNA-seq绝对定量UMI测序技术
为了解决现有RNA相关测序技术的技术难点，获得更准确的表达数据，康测科技开发出“绝对定量测序”技术。
该技术在文库扩增之前为每一条逆转录的cDNA片段加上唯一的身份标签（Unique Identifier，UMI），也称为数字标签，数字标签会伴随片段扩增、测序、分析的全过程。
绝对定量测序建库完成之后，同一个片段经过PCR扩增出来的产物均带有相同的数字标签，测序完成后利用UMI追溯每一个片段的来源，将相同来源的片段（具有相同的序列和UMI）进行合并，就能准确去除PCR扩增重复，一比一准确还原样本扩增前的原始状态。在此过程中，PCR扩增和测序错误同样可以被纠正：扩增和测序的错误会使得相同UMI标签对应多个不同的序列，那么只需比较这些序列的相似性，即可纠正这些错误。
康测科技RNA相关测序产品，全线采用了KC-digital RNA-seq绝对定量UMI测序技术
技术优势
实测数据
UMI可以消除PCR扩增偏好引入的结果差异
左图：使用普通RNA建库试剂盒对同一个样品在不同扩增条件下获得的基因表达的一致性；
右图：使用KC-digitalRNA建库试剂盒对同一个样品在不同扩增条件下获得的基因表达的一致性。
KC-digital RNA-seq带来更高的qPCR验证结果
选择48个差异表达基因进行qPCR验证，分析qPCR和测序结果的相关性
左图：普通RNA-seq与qPCR实验结果的比较；右图：KC-Digital RNA-seq与qPCR实验结果的比较；
KC-DigitalRNA-seq测序结果与qPCR结果更为相符，有更高的一致性。
部分客户文章（绝对定量转录组）
- Wu J, Wu C, Xing F, et al. Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells[J]. Cell Research, 2021: 1-13. (IF:20.507)
- Fei Xie , Chao Huang, et al. CircPTPRA blocks the recognition of RNA N6 -methyladenosine through interacting with IGF2BP1 to suppress bladder cancer progression[J].Molecular Cancer., 2021. （IF：15.302）
- Zhang Min,Wang Peng,Luo Ronghua,Wang Yaqing,Li Zhongyu,Guo Yaqiong,Yao Yulin,Li Minghua,Tao Tingting,Chen Wenwen,Han Jianbao,Liu Haitao,Cui Kangli,Zhang Xu,Zheng Yongtang,Qin Jianhua. Biomimetic Human Disease Model of SARS‐CoV‐2‐Induced Lung Injury and Immune Responses on Organ Chip System[J]. Advanced Science,2020,8(3). （IF：15.840）
- Huang Z, Chu L, Liang J, et al. H19 Promotes HCC Bone Metastasis Through Reducing OPG Expression in a PPP1CA/p38MAPK‐Dependent Manner and Sponging miR‐200b‐3p[J]. Hepatology, 2020. （IF：14.679）
- Yi Ming,Zhang Jing,Li Anping,Niu Mengke,Yan Yongxiang,Jiao Ying,Luo Suxia,Zhou Pengfei,Wu Kongming. The construction, expression, and enhanced anti-tumor activity of YM101: a bispecific antibody simultaneously targeting TGF-β and PD-L1.[J]. Journal of hematology & oncology,2021,14(1). （IF：11.059）
- Guo Yaqiong,Luo Ronghua,Wang Yaqing,Deng Pengwei,Song Tianzhang,Zhang Min,Wang Peng,Zhang Xu,Cui Kangli,Tao Tingting,Li Zhongyu,Chen Wenwen,Zheng Yongtang,Qin Jianhua. SARS-CoV-2 induced intestinal responses with a biomimetic human gut-on-chip[J]. Science Bulletin,2020(prepublish). （IF:9.511）
- Feng Liu,Hui Zhang, Fei Xie,Dan Tao, Xing yuan Xiao, Chao Huang, Miao Wang,Chaohui Gu,Xiaoping Zhang,Guosong Jiang. Hsa_circ_0001361 promotes bladder cancer invasion and metastasis through miR-491-5p/MMP9 axis.F Liu et al. Oncogene (2020) （IF：6.634）
- Yang L, Zhou X, Sun J, et al. Reactive oxygen species mediate anlotinib-induced apoptosis via activation of endoplasmic reticulum stress in pancreatic cancer[J]. Cell Death & Disease. (2020)（IF：6.39）
- Song Chao,Shi Dongmei,Chang Kaiwen,Li Xianghui,Dong Qing,Ma Xia,Wang Xuefei,Guo Zhenhuan,Liu Yonglu,Wang Jundong. Sodium fluoride activates the extrinsic apoptosis via regulating NOX4/ROS-mediated p53/DR5 signaling pathway in lung cells both in vitro and in vivo[J]. Free Radical Biology and Medicine,2021,169.（IF：6.170）
- Zhu Z, Pan Q, Zhao W, Wu X, Yu S, Shen Q, Zhang J, Yue W, Peng S, Li N, Zhang S, Lei A, Hua J. BCL2 enhances survival of porcine pluripotent stem cells through promoting FGFR2. Cell Prolif. 2021 Jan;54(1):e12932. doi: 10.1111/cpr.12932. Epub 2020 Oct 26. PMID: 33107129; PMCID: PMC7791183. （IF：5.753）
- Ning Sun, Xinping Zhong, Shengli Wang, Kai Zeng, Hongmiao Sun, Ge Sun, Renlong Zou, Wei Liu, Wensu Liu, Lin Lin, Huijuan Song, Chi Lv, Chunyu Wang, Yue Zhao,ATXN7L3 positively regulates SMAD7 transcription in hepatocellular carcinoma with growth inhibitory function,EBioMedicine,2020. （IF：5.736）
- Chen Wenwen,Cao Rongkai,Su Wentao,Zhang Xu,Xu Yuhai,Wang Peng,Gan Zhongqiao,Xie Yingying,Li Hongjing,Qin Jianhua. Simple and fast isolation of circulating exosomes with a chitosan modified shuttle flow microchip for breast cancer diagnosis.[J]. Lab on a chip,2021.（IF：6.774）
- Yang Liguo,Zhou Xiaoshu,Sun Jinrui,Lei Qianghui,Wang Qi,Pan Di,Ding Mingxing,Ding Yi. Reactive oxygen species mediate anlotinib-induced apoptosis via activation of endoplasmic reticulum stress in pancreatic cancer.[J]. Cell death & disease,2020,11(9). （IF：6.39）
- Qu Z, Fu Y, Lin Y, Zhao Z, Zhang X, Cheng J, Xie J, Chen T, Li B, Jiang D. Transcriptional Responses of Sclerotinia sclerotiorum to the Infection by SsHADV-1. Journal of Fungi. 2021; 7(7):493.（IF:5.340，一区）
- Zhu Z , Wu X , Li Q , et al. Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to construct pluripotency gene regulatory network. 2020. （IF：4.966）
- Shen Shiyi and Wei Chunli and Fu Junjiang. RNA-Sequencing Reveals Heat Shock 70-kDa Protein 6 (HSPA6) as a Novel Thymoquinone-Upregulated Gene That Inhibits Growth, Migration, and Invasion of Triple-Negative Breast Cancer Cells [J]. Frontiers in Oncology, 2021. （IF：4.848）
- Xue W, Zhang J, Tong H, et al. Effects of BMPER, CXCL10, and HOXA9 on Neovascularization During Early-Growth Stage of Primary High-Grade Glioma and Their Corresponding MRI Biomarkers[J]. Frontiers in Oncology, 2020, 10:711. （IF：4.848）
- Wei guang Kong, Yong-yao Yu, Shuai Dong, Zhen-yuHuang, Li-guo Ding, Jia-feng Cao, Fen Dong, Xiao ting Zhang, Xia Liu, Hao-yue Xu, Kai-feng Meng, Jian-guo Su and Zhen Xu. Pharyngeal Immunity in Early Vertebrates Provides Functional and Evolutionary Insight into Mucosal Homeostasis.WG Kong et al. J Immunol (2019) （IF：4.718）
- Fan W A , Qs A , Qx A , et al. A bioinformatics and transcriptomics based investigation reveals an inhibitory role of Huanglian-Renshen-Decoction on hepatic glucose production of T2DM mice via PI3K/Akt/FoxO1 signaling pathway - ScienceDirect[J]. Phytomedicine, 2021. （IF：4.268）
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文献和实验该产品被引用文献
部分客户应用文章发表示例（绝对定量转录组测序）
- Wu J, Wu C, Xing F, et al. Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells[J]. Cell Research, 2021: 1-13. (IF:20.507)
- Fei Xie , Chao Huang, et al. CircPTPRA blocks the recognition of RNA N6 -methyladenosine through interacting with IGF2BP1 to suppress bladder cancer progression[J].Molecular Cancer., 2021. （IF：15.302）
- Zhang Min,Wang Peng,Luo Ronghua,Wang Yaqing,Li Zhongyu,Guo Yaqiong,Yao Yulin,Li Minghua,Tao Tingting,Chen Wenwen,Han Jianbao,Liu Haitao,Cui Kangli,Zhang Xu,Zheng Yongtang,Qin Jianhua. Biomimetic Human Disease Model of SARS‐CoV‐2‐Induced Lung Injury and Immune Responses on Organ Chip System[J]. Advanced Science,2020,8(3). （IF：15.840）
- Huang Z, Chu L, Liang J, et al. H19 Promotes HCC Bone Metastasis Through Reducing OPG Expression in a PPP1CA/p38MAPK‐Dependent Manner and Sponging miR‐200b‐3p[J]. Hepatology, 2020. （IF：14.679）
- Yi Ming,Zhang Jing,Li Anping,Niu Mengke,Yan Yongxiang,Jiao Ying,Luo Suxia,Zhou Pengfei,Wu Kongming. The construction, expression, and enhanced anti-tumor activity of YM101: a bispecific antibody simultaneously targeting TGF-β and PD-L1.[J]. Journal of hematology & oncology,2021,14(1). （IF：11.059）
- Guo Yaqiong,Luo Ronghua,Wang Yaqing,Deng Pengwei,Song Tianzhang,Zhang Min,Wang Peng,Zhang Xu,Cui Kangli,Tao Tingting,Li Zhongyu,Chen Wenwen,Zheng Yongtang,Qin Jianhua. SARS-CoV-2 induced intestinal responses with a biomimetic human gut-on-chip[J]. Science Bulletin,2020(prepublish). （IF:9.511）
- Feng Liu,Hui Zhang, Fei Xie,Dan Tao, Xing yuan Xiao, Chao Huang, Miao Wang,Chaohui Gu,Xiaoping Zhang,Guosong Jiang. Hsa_circ_0001361 promotes bladder cancer invasion and metastasis through miR-491-5p/MMP9 axis.F Liu et al. Oncogene (2020) （IF：6.634）
- Yang L, Zhou X, Sun J, et al. Reactive oxygen species mediate anlotinib-induced apoptosis via activation of endoplasmic reticulum stress in pancreatic cancer[J]. Cell Death & Disease. (2020)（IF：6.39）
- Song Chao,Shi Dongmei,Chang Kaiwen,Li Xianghui,Dong Qing,Ma Xia,Wang Xuefei,Guo Zhenhuan,Liu Yonglu,Wang Jundong. Sodium fluoride activates the extrinsic apoptosis via regulating NOX4/ROS-mediated p53/DR5 signaling pathway in lung cells both in vitro and in vivo[J]. Free Radical Biology and Medicine,2021,169.（IF：6.170）
- Zhu Z, Pan Q, Zhao W, Wu X, Yu S, Shen Q, Zhang J, Yue W, Peng S, Li N, Zhang S, Lei A, Hua J. BCL2 enhances survival of porcine pluripotent stem cells through promoting FGFR2. Cell Prolif. 2021 Jan;54(1):e12932. doi: 10.1111/cpr.12932. Epub 2020 Oct 26. PMID: 33107129; PMCID: PMC7791183. （IF：5.753）
- Ning Sun, Xinping Zhong, Shengli Wang, Kai Zeng, Hongmiao Sun, Ge Sun, Renlong Zou, Wei Liu, Wensu Liu, Lin Lin, Huijuan Song, Chi Lv, Chunyu Wang, Yue Zhao,ATXN7L3 positively regulates SMAD7 transcription in hepatocellular carcinoma with growth inhibitory function,EBioMedicine,2020. （IF：5.736）
- Chen Wenwen,Cao Rongkai,Su Wentao,Zhang Xu,Xu Yuhai,Wang Peng,Gan Zhongqiao,Xie Yingying,Li Hongjing,Qin Jianhua. Simple and fast isolation of circulating exosomes with a chitosan modified shuttle flow microchip for breast cancer diagnosis.[J]. Lab on a chip,2021.（IF：6.774）
- Yang Liguo,Zhou Xiaoshu,Sun Jinrui,Lei Qianghui,Wang Qi,Pan Di,Ding Mingxing,Ding Yi. Reactive oxygen species mediate anlotinib-induced apoptosis via activation of endoplasmic reticulum stress in pancreatic cancer.[J]. Cell death & disease,2020,11(9). （IF：6.39）
- Qu Z, Fu Y, Lin Y, Zhao Z, Zhang X, Cheng J, Xie J, Chen T, Li B, Jiang D. Transcriptional Responses of Sclerotinia sclerotiorum to the Infection by SsHADV-1. Journal of Fungi. 2021; 7(7):493.（IF:5.340，一区）
- Zhu Z , Wu X , Li Q , et al. Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to construct pluripotency gene regulatory network. 2020. （IF：4.966）
- Shen Shiyi and Wei Chunli and Fu Junjiang. RNA-Sequencing Reveals Heat Shock 70-kDa Protein 6 (HSPA6) as a Novel Thymoquinone-Upregulated Gene That Inhibits Growth, Migration, and Invasion of Triple-Negative Breast Cancer Cells [J]. Frontiers in Oncology, 2021. （IF：4.848）
- Xue W, Zhang J, Tong H, et al. Effects of BMPER, CXCL10, and HOXA9 on Neovascularization During Early-Growth Stage of Primary High-Grade Glioma and Their Corresponding MRI Biomarkers[J]. Frontiers in Oncology, 2020, 10:711. （IF：4.848）
- Wei guang Kong, Yong-yao Yu, Shuai Dong, Zhen-yuHuang, Li-guo Ding, Jia-feng Cao, Fen Dong, Xiao ting Zhang, Xia Liu, Hao-yue Xu, Kai-feng Meng, Jian-guo Su and Zhen Xu. Pharyngeal Immunity in Early Vertebrates Provides Functional and Evolutionary Insight into Mucosal Homeostasis.WG Kong et al. J Immunol (2019) （IF：4.718）
- Fan W A , Qs A , Qx A , et al. A bioinformatics and transcriptomics based investigation reveals an inhibitory role of Huanglian-Renshen-Decoction on hepatic glucose production of T2DM mice via PI3K/Akt/FoxO1 signaling pathway - ScienceDirect[J]. Phytomedicine, 2021. （IF：4.268）
- Sitong Liu,Yijun Tang,Nan Ruan,Zhengjun Dang,Yuwei Huang,Wei Miao,Zhengjin Xu,Fengcheng Li. The Rice BZ1 Locus Is Required for Glycosylation of Arabinogalactan Proteins and Galactolipid and Plays a Role in both Mechanical Strength and Leaf Color[J]. Rice,2020,13(1).（IF：3.912）
- Xiang Taihe,Li Jiangshan,Bao Shuying,Xu Zhengxian,Wang Leizhen,Long Fazong,He Chenjing. Digital RNA-seq transcriptome plus tissue anatomy analyses reveal the developmental mechanism of the calabash-shaped root in Tetrastigma hemsleyanum.[J]. Tree physiology,2021.（IF：3.655）
- Sun M, Li H, Li Y, et al. Tomato YABBY2b controls plant height through regulating indole-3-acetic acid-amido synthetase (GH3.8) expression[J]. Plant Science, 2020, 297:110530. （IF：3.4）
- Wang Limin,Xie Shunpei,Zhang Yinshan,Kang Ruijiao,Zhang Mengjuan,Wang Min,Li Haiyang,Chen Linlin,Yuan Hongxia,Ding Shengli,Liang Shen,Li Honglian. The FpPPR1 Gene Encodes a Pentatricopeptide Repeat Protein That Is Essential for Asexual Development, Sporulation, and Pathogenesis in Fusarium pseudograminearum [J]. Frontiers in Genetics,2021. （IF：3.258）
- Wang Zhijie,Ji Shangli,Huang Yinhua,Liao Kai,Cui Zekai,Chu Feixue,Chen Jiansu,Tang Shibo. The daily gene transcription cycle in mouse retina[J]. Experimental Eye Research,2021(prepublish).（IF：3.011）
- Wu M , Deng Q , Lei X , et al. Elavl2 Regulates Retinal Function Via Modulating the Differentiation of Amacrine Cells Subtype[J]. Investigative Ophthalmology & Visual Science, 2021, 62(7):1. （IF：3.0）
- Yong Yao Yu, Wei-Guang Kong, Hao-Yue Xu, Zhen-Yu Huang, Xiao-Ting Zhang, Li-Guo Ding, Shuai Dong, Guang-Mei Yin, Fen Dong, Wei Yu, Jia-Feng Cao, Kai-Feng Meng, Xia Liu, Yu Fu, Xue-zhen Zhang, Yong-an Zhang, J. Oriol Sunyer, and Zhen Xu. Convergent Evolution of Mucosal Immune Responses at the Buccal Cavity of Teleost Fish.YY Yu et al. iScience (2019) （IF：2.1）
- Wang Zhi,Dong Hui,Yang Li,Yi Ping,Wang Qing,Huang Dongmei. The role of FDX1 in granulosa cell of Polycystic ovary syndrome (PCOS).[J]. BMC endocrine disorders,2021,21(1).（IF:1.994）
- Li Y, Zhang Q, Ou L, et al. Response to the Cold Stress Signaling of the Tea Plant (Camellia sinensis) Elicited by Chitosan Oligosaccharide[J]. 2020.（IF：1.614）
相关实验
结合细胞在组织中分布的位置信息和转录组基因表达信息，了解细胞在空间背景下的异质性。
研究意义；而同样情况下RASGRP1的检测数据可能不能说明问题。 由此可知，设计的实验如果没有生物学重复，或者生物学重复的数量不够，就不能得到有统计意义的实验结果；获得的差异表达的基因很可能仅仅是少数个体差异的表现，并不能反映疾病或者某种特定生理状态的群体本质特征。 3.生物学重复设置几个合适？ 您是不是有同样的问题：转录组测序是否必须进行生物学重复啊，是否要3个重复，是否可以用3个样品的RNA等量混合代替生物学重复，如果不重复能否发文章…..？一方面是有限的经费，一方面是编辑的质疑；实在很难
其序列和结构影响转录的延伸、暂停和停顿，以及发挥染色体修饰结合和增强子的作用。nascent RNA- seq方法旨在区分新近转录的RNA和其它RNAs，这些方法可以分为4类：溴尿苷(BrU)免疫共沉淀深度测序分析（ BRIC-seq ），4sU-seq，瞬时转录组测序（ TT-seq），代谢标记法（SLAM-seq）(图2)。
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绝对定量转录组测序（Digital RNA-seq）
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