1、 1 目 录 RNAi 及基因沉默原理 百奥迈科（ Biomics） RNAi 产品与服务一览 1.siRNA 生物合成 1.1 全位点 siRNA 表达文库构建服务 1.2 RNAi 载体构建技术服务 1.3 Gene TargetTMsiRNA Set 2.siRNA 化学合成 2.1 化学合成 siRNA 简介 2.2 siRNA 设计 2.3 百奥迈科 (Biomics) siRNA 产品简介 2.4 普通 siRNA oligo 2.5 荧光标记 siRNA oligo 2.6 化学修饰 siRNA oligo 2.7 miRNA 的化学合成 2.8 RNAi 系列优惠套餐 2.9
2、siRNA 对照 3.RNAi 病毒包装 3.1 腺病毒载体包装 3.2 慢病毒载体包装 3.3 其他类型病毒载体包装 4.哺乳动物细胞 siRNA 转染 4.1 转染方法 4.2 转染步骤 4.3 转染优化及注意事项 5.mRNA 水平检测基因沉默 5.1 mRNA 的提取 /制备 5.2. 实时定量荧光 PCR 6 蛋白质水平检测基因沉默 6.1 Western blot 6.2 ELISA 6.3 免疫荧光 7.其他相关服务 7.1 稳定细胞筛选服务 7.2 细胞增殖毒性检测服务 7.3 细胞凋亡检测服务 7.4 DNA 测序 8.RNAi 相关产品 8.1 RISOTMRNA 抽提试剂
3、 8.2 其他 2 RNAi 及基因沉默原理 RNA 干扰 (RNAinterference， RNAi)是由双链 RNA (double-strandedRNA， dsRNA) 引发的转录后基因静默机制。其原理是： RNaseIII 核酶家族的 Dicer，与双链 RNA 结合，将其剪切成 21 - 25nt 及 3端突出的小干扰 RNA (small interfering RNA， siRNA)，随后 siRNA 与 RNA诱导沉默复合物 (RNA - induced silencing complex， RISC) 结合，解旋成单链，活化的 RISC受已成单链的 siRNA 引导，序列
4、特异性地结合在靶 mRNA 上并将其切断，引发靶 mRNA 的特异性分解，从而阻断相应基因表达的转录后基因沉默机制。 3 百奥迈科（ Biomics） RNAi 产品与服务一览 4 1. siRNA 生物合成 1.1 全位点 siRNA 表达文库构建服务 本公司拥有自主知识产权的全位点 siRNA 文库构建技术，能够从全长 cDNA 构建包含所有有效结合位点，长度分布于 19 - 23bp 的靶基因的 siRNA 分子库，亦可以模仿体内 Dicer 酶切割长度为 21 - 23bp 的分子库，适用于高通量功能基因及相关药物靶点筛选。 1.1.1 构建流程： 1）客户可选： z 提供靶基因的 c
5、DNA 克隆。 z 提供靶基因的相关信息 (基因的 mRNA 序列或 Genbank Accession Number)。 z 也可在本公司的 cDNA 产品目录中，挑选靶基因 cDNA 克隆。 2）进行 siRNA 文库构建，抽样测序验证。 3）根据客户要求提供相应的文库产品。 4）将其连同抽样报告、产品说明书一起寄送到客户手中。 A. HBV Polymerase Domain siRNA 表达文库位点分布（随机抽样测序） Polymerase Domain (984bp): B. 构建的 Survivin 基因全位点 siRNA 表达文库，文库抽样基因沉默 (qPCR)筛选结果： 该数据
6、显示，本公司 siRNA 文库位点分布的随机性，可以更好地找到最优的靶点。 s1-s8: Randomly selected siRNA sequences from Biomics library s9/s10/s11: The published siRNA sequences target-off: Negative control un-transfect: Untreated normal control 5 1.2 RNAi 载体构建技术服务 1.2.1 技术特点： 1）多种 siRNA 表达载体，可以在细胞内直接转录出 siRNA，行使其 RNAi 功能，包括： z U6 启动子
7、表达载体； z H1 启动子表达载体； z U6、 H1 双启动子表达载体； z CMV 启动子表达载体； 2）载体带有 GFP 标签可以监测转染效率。构建好的 siRNA 表达载体可直接用于转染细胞进行 RNAi 实验 ; 3）本公司还可根据客户需要提供各种商业化表达载体 ; 4）本公司可以为客户构建线性 siRNA 表达载体， shRNA 表达载体， miRNA 表达载体以及病毒型 RNAi 载体。 1.2.2 构建流程： 1） siRNA 靶点设计； z 客户提供靶基因的 siRNA 序列，由本公司将其克隆到 siRNA 表达载体上。 z 客户提供靶基因的相关信息（基因的 mRNA 序列
8、或 Genbank Accession No.） ，根据客户要求，本公司设计高效特异性的 3-4 条针对靶基因的 siRNA。 2）合成设计好的 DNA 并退火形成双链 DNA，克隆到客户要求的载体上。 3）通过测序进行验证。 4）构建成功的 siRNA 质粒连同测序报告、产品说明书一起寄送到客户手中。 注：可根据客户要求提供 siRNA 的阳性及阴性对照质粒。 1.2.3 质量保证： 符合设计要求的 siRNA 序列测序结果。 基于载体的 RNAi 实验示意图 6 1.2.4 各种商业化表达载体 : 产品编号 载体 启动子 筛选基因 标记基因 BV0011 pRNAi-U6H1/Neo U6
9、 和 H1 Neomycin GFP BV0012 pRNAi-H1.1/Neo H1 Neomycin GFP BV0013 pRNAi-U6.1/Neo U6 Neomycin GFP BV0014 pRNAi-CMV3.1/Neo CMV Neomycin GFP BV0015 pRNAi-CMV3.2/Neo CMV Neomycin GFP BV0016 pRNAi-CMV4.1/Neo CMV Neomycin - BV0107 pRNAi-U6.1/Lenti U6 Neomycin GFP BV0108 pRNAi-H1.1/Retro H1 Neomycin GFP 7 1.
10、3. Gene TargetTMsiRNA Set Gene Target siRNA Set 是本公司的专利技术开发的基因沉默系列产品之一。产品由 4个不同位点的 siRNA 克隆组成，可以降低在基因沉默中产生的脱靶现象，提高沉默效率。四个克隆可以混合使用，也可以单独使用。产品均经基因测序证实，保证其 100%的同源性。 本产品有如下特点： z 在 U6 和 H1 启动子的起始序列 AAAAA 和终止序列 TTTTT 之间的 siRNA 片段没有多余的多克隆位点序列，提高沉默的特异性。 z siRNA 表达框（ U6 siRNA H1）可以整体亚克隆到逆转录病毒载体 (Retrovirus)
11、、腺病毒载体（ Adenovirus） 、慢病毒载体（ Lentivirus） ，具有较好的适用性。 z siRNA 没有二级结构 (如发夹型结构 )，在上述病毒载体中较稳定。 z 构建的质粒载体转染到细胞内，在细胞内转录出双链 RNA，在细胞内具有中长期表达的特性，能够较长期的发挥基因沉默的作用。 z 由于质粒可以复制扩增，较化学合成方法显著降低制备 siRNA 的成本。 z 载体法的 siRNA 可测序保证 100%序列同源性，而化学合成的 siRNA 分子的序列鉴定较困难。 表达载体 pU6H1-GFP 图谱 产品性状形态：本产品具有可靠的稳定性，以质粒 DNA 与滤纸小圆片共干燥封装，
12、可在常温下运输和储存。 Gene TargetTMsiRNA Set 产品包含： 1. 特定基因 4 个位点的 Gene TargetTMsiRNA Clone。 2. 阳性对照 1 管（用于平行跟踪转染、 RNA 提取等实验过程，排除假阴性结果。 ） 3. 阴性对照 1 管 （阴性对照的 siRNA 与目的基因以及靶细胞中其它基因 mRNA没有同源性， 排除假阳性结果。 ） 8 Gene TargetTMsiRNA Set 产品目录 Cat.No. ACCN No. Description Kinase K07002 NM_145262 Homo sapiens glycerate kina
13、se (GLYCTK) K07003 NM_145259 Homo sapiens activin A receptor, type IC (ACVR1C) K07004 NM_153361 Homo sapiens hypothetical protein MGC42105 (MGC42105) K07005 NM_153273 Homo sapiens inositol hexaphosphate kinase 1 (IHPK1), transcript variant 1 K07007 NM_152720 Homo sapiens NIMA (never in mitosis gene
14、a)-related kinase 3 (NEK3) K07008 NM_030906 Homo sapiens serine/threonine kinase 33 (STK33) K07009 NM_020804 Homo sapiens protein kinase C and casein kinase substrate in neurons 1 (PACSIN1) K07012 NM_172170 Homo sapiens calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma (CAMK2G), tran
15、script variant 3 K07016 NM_152835 Homo sapiens PDLIM1 interacting kinase 1 like (PDIK1L) K07021 NM_015000 Homo sapiens serine/threonine kinase 38 like (STK38L) K07026 NM_174858 Homo sapiens adenylate kinase 5 (AK5), transcript variant 1 K07027 NM_173354 Homo sapiens SNF1-like kinase (SNF1LK) K07029
16、NM_033015 Homo sapiens Fas-activated serine/threonine kinase (FASTK), transcript variant 4 K07030 NM_032844 Homo sapiens microtubule associated serine/threonine kinase-like (MASTL) K07033 BC032542 Homo sapiens, L-fucose kinase, clone MGC:45494 IMAGE:5534376, complete cds K07035 NM_032028 Homo sapien
17、s testis-specific serine kinase 1 (TSSK1) K07041 NM_031414 Homo sapiens serine/threonine kinase 31 (STK31), transcript variant 1 K07046 NM_182691 Homo sapiens SFRS protein kinase 2 (SRPK2), transcript variant 2 K07054 NM_032409 Homo sapiens PTEN induced putative kinase 1 (PINK1), nuclear gene encodi
18、ng mitochondrial protein K07055 NM_023018 Homo sapiens NAD kinase (NADK) K07056 NM_024876 Homo sapiens aarF domain containing kinase 4 (ADCK4) K07057 NM_006153 Homo sapiens NCK adaptor protein 1 (NCK1) K07059 NM_006293 Homo sapiens TYRO3 protein tyrosine kinase (TYRO3) 9 K07060 NM_024761 Homo sapien
19、s MOB1, Mps One Binder kinase activator-like 2B (yeast) (MOBKL2B) K07061 NM_173515 Homo sapiens CNKSR family member 3 (CNKSR3) K07066 NM_002497 Homo sapiens NIMA (never in mitosis gene a)-related kinase 2 (NEK2) Gene TargetTMsiRNA Set 产品目录 Cat.No. ACCN No. Description K07067 NM_002350 Homo sapiens v
20、-yes-1 Yamaguchi sarcoma viral related oncogene homolog (LYN) K07069 NM_002731 Homo sapiens protein kinase, cAMP-dependent, catalytic, beta (PRKACB), K07071 NM_020421 Homo sapiens aarF domain containing kinase 1 (ADCK1) K07076 NM_171825 Homo sapiens calcium/calmodulin-dependent protein kinase (CaM k
21、inase) II alpha (CAMK2A), K07079 NM_001798 Homo sapiens cyclin-dependent kinase 2 (CDK2), transcript variant 1 K07080 NM_004936 Homo sapiens cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (CDKN2B), transcript variant 1 K07082 NM_003992 Homo sapiens CDC-like kinase 3 (CLK3), K07085 NM_0052
22、33 Homo sapiens EPH receptor A3 (EPHA3), transcript variant 1 K07087 NM_004103 Homo sapiens PTK2B protein tyrosine kinase 2 beta (PTK2B), transcript variant 2 K07089 NM_000801 Homo sapiens FK506 binding protein 1A, 12kDa (FKBP1A), transcript variant 12B K07090 NM_032999 Homo sapiens general transcri
23、ption factor II, i (GTF2I), transcript variant 1 K07091 NM_030952 Homo sapiens NUAK family, SNF1-like kinase, 2 (NUAK2) K07092 NM_000221 Homo sapiens ketohexokinase (fructokinase) (KHK), transcript variant a K07093 NM_002498 Homo sapiens NIMA (never in mitosis gene a)-related kinase 3 (NEK3), K07096
24、 NM_181839 Homo sapiens protein kinase (cAMP-dependent, catalytic) inhibitor alpha (PKIA), transcript variant 7 K07100 NM_145160 Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant A K07102 NM_002822 Homo sapiens twinfilin, actin-binding protein, homolog 1 (Drosophila
25、) (TWF1) K07105 NM_003583 Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2), transcript variant 1 K07108 NM_003668 Homo sapiens mitogen-activated protein kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 1 K07109 NM_139078 Homo sapiens mitogen-activ
26、ated protein kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 2 K07110 NM_030662 Homo sapiens mitogen-activated protein kinase kinase 2 (MAP2K2) 10 K07111 NM_025233 Homo sapiens Coenzyme A synthase (COASY), transcript variant 1 K07112 NM_170693 Homo sapiens serum/glucocorticoid regul
27、ated kinase 2 (SGK2), transcript variant 1 K07116 NM_007194 Homo sapiens CHK2 checkpoint homolog (S. pombe) (CHEK2), transcript variant 1 K07118 NM_017593 Homo sapiens BMP2 inducible kinase (BMP2K), transcript variant 2 K07121 NM_004336 Homo sapiens BUB1 budding uninhibited by benzimidazoles 1 homol
28、og (yeast) (BUB1) K07122 NM_005308 Homo sapiens G protein-coupled receptor kinase 5 (GRK5) Gene TargetTMsiRNA Set 产品目录 Cat.No. ACCN No. Description K07125 NM_002648 Homo sapiens pim-1 oncogene (PIM1) K07126 NM_005030 Homo sapiens polo-like kinase 1 (Drosophila) (PLK1) K07128 NM_006374 Homo sapiens s
29、erine/threonine kinase 25 (STE20 homolog, yeast) (STK25) K07130 NM_021643 Homo sapiens tribbles homolog 2 (Drosophila) (TRIB2) K07131 NM_013254 Homo sapiens TANK-binding kinase 1 (TBK1) K07132 NM_020247 Homo sapiens chaperone, ABC1 activity of bc1 complex homolog (S. pombe) (CABC1), nuclear gene enc
30、oding mitochondrial protein K07133 NM_144685 Homo sapiens homeodomain interacting protein kinase 4 (HIPK4) K07136 NM_002752 Homo sapiens mitogen-activated protein kinase 9 (MAPK9), transcript variant JNK2-a2 K07137 NM_024046 Homo sapiens CaM kinase-like vesicle-associated (CAMKV) K07138 NM_021913 Ho
31、mo sapiens AXL receptor tyrosine kinase (AXL), transcript variant 1 K07141 NM_021135 Homo sapiens ribosomal protein S6 kinase, 90kDa, polypeptide 2 (RPS6KA2), transcript variant 1 K07142 NM_021158 Homo sapiens tribbles homolog 3 (Drosophila) (TRIB3) K07143 NM_000075 Homo sapiens cyclin-dependent kin
32、ase 4 (CDK4) K07144 NM_020168 Homo sapiens p21(CDKN1A)-activated kinase 6 (PAK6) K07145 NM_018571 Homo sapiens amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 2 (ALS2CR2) K07146 NM_018492 Homo sapiens PDZ binding kinase (PBK) K07148 NM_018401 Homo sapiens serine/threonine kin
33、ase 32B (STK32B) 11 K07150 NM_018343 Homo sapiens RIO kinase 2 (yeast) (RIOK2) K07151 NM_017988 Homo sapiens SCY1-like 2 (S. cerevisiae) (SCYL2) K07152 NM_017719 Homo sapiens SNF related kinase (SNRK) K07154 NM_016281 Homo sapiens TAO kinase 3 (TAOK3) K07155 NM_016123 Homo sapiens interleukin-1 rece
34、ptor-associated kinase 4 (IRAK4) K07158 NM_014683 Homo sapiens unc-51-like kinase 2 (C. elegans) (ULK2) K07160 NM_014413 Homo sapiens eukaryotic translation initiation factor 2-alpha kinase 1 (EIF2AK1) K07161 NM_014365 Homo sapiens heat shock 22kDa protein 8 (HSPB8) K07162 NM_014264 Homo sapiens pol
35、o-like kinase 4 (Drosophila) (PLK4) Gene TargetTMsiRNA Set 产品目录 Cat.No. ACCN No. Description K07163 NM_013257 Homo sapiens serum/glucocorticoid regulated kinase family, member 3 (SGK3), transcript variant 1 K07164 NM_007284 Homo sapiens twinfilin, actin-binding protein, homolog 2 (Drosophila) (TWF2)
36、 K07166 NM_007199 Homo sapiens interleukin-1 receptor-associated kinase 3 (IRAK3) K07168 NM_006712 Homo sapiens Fas-activated serine/threonine kinase (FASTK), transcript variant 1 K07169 NM_006622 Homo sapiens polo-like kinase 2 (Drosophila) (PLK2) K07170 NM_006281 Homo sapiens serine/threonine kina
37、se 3 (STE20 homolog, yeast) (STK3) K07171 NM_006285 Homo sapiens testis-specific kinase 1 (TESK1) K07172 NM_006296 Homo sapiens vaccinia related kinase 2 (VRK2) K07173 NM_006296 VARIANT 1 of Homo sapiens vaccinia related kinase 2 (VRK2) K07174 NM_006252 Homo sapiens protein kinase, AMP-activated, al
38、pha 2 catalytic subunit (PRKAA2) K07175 NM_212539 Homo sapiens protein kinase C, delta (PRKCD), transcript variant 2 K07176 NM_006255 Homo sapiens protein kinase C, eta (PRKCH) K07177 NM_006213 Homo sapiens phosphorylase kinase, gamma 1 (muscle) (PHKG1) K07178 NM_006182 Homo sapiens discoidin domain
39、 receptor family, member 2 (DDR2), transcript variant 2 K07179 NM_005990 Homo sapiens serine/threonine kinase 10 (STK10) 12 K07180 NM_005923 Homo sapiens mitogen-activated protein kinase kinase kinase 5 (MAP3K5) K07181 NM_005881 Homo sapiens branched chain ketoacid dehydrogenase kinase (BCKDK) K0718
40、2 NM_005762 Homo sapiens tripartite motif-containing 28 (TRIM28) K07183 NM_005627 Homo sapiens serum/glucocorticoid regulated kinase (SGK) K07185 NM_000020 Homo sapiens activin A receptor type II-like 1 (ACVRL1), transcript variant 1 K07186 NM_005433 Homo sapiens v-yes-1 Yamaguchi sarcoma viral onco
41、gene homolog 1 (YES1) K07188 NM_005211 Homo sapiens colony stimulating factor 1 receptor, formerly McDonough feline sarcoma viral (v-fms) oncogene homolog (CSF1R) K07190 NM_005044 Homo sapiens protein kinase, X-linked (PRKX) K07192 NM_004760 Homo sapiens serine/threonine kinase 17a (apoptosis-induci
42、ng) (STK17A) K07194 NM_004517 Homo sapiens integrin-linked kinase (ILK), transcript variant 1 K07195 NM_004443 Homo sapiens EPH receptor B3 (EPHB3) Gene TargetTMsiRNA Set 产品目录 Cat.No. ACCN No. Description K07197 NM_004409 Homo sapiens dystrophia myotonica-protein kinase (DMPK) K07198 NM_004226 Homo
43、sapiens serine/threonine kinase 17b (apoptosis-inducing) (STK17B) K07200 NM_004071 Homo sapiens CDC-like kinase 1 (CLK1), transcript variant 1 K07201 NM_004073 Homo sapiens polo-like kinase 3 (Drosophila) (PLK3) K07202 NM_003952 Homo sapiens ribosomal protein S6 kinase, 70kDa, polypeptide 2 (RPS6KB2
44、), K07203 NM_003954 Homo sapiens mitogen-activated protein kinase kinase kinase 14 (MAP3K14) K07204 NM_003821 Homo sapiens receptor-interacting serine-threonine kinase 2 (RIPK2) K07208 NM_003318 Homo sapiens TTK protein kinase (TTK) K07210 NM_003177 Homo sapiens spleen tyrosine kinase (SYK) K07211 N
45、M_003188 Homo sapiens mitogen-activated protein kinase kinase kinase 7 (MAP3K7), transcript variant A K07212 NM_003137 Homo sapiens SFRS protein kinase 1 (SRPK1) K07214 NM_003010 Homo sapiens mitogen-activated protein kinase kinase 4 (MAP2K4) 13 K07219 NM_002969 Homo sapiens mitogen-activated protei
46、n kinase 12 (MAPK12) K07221 NM_002880 Homo sapiens v-raf-1 murine leukemia viral oncogene homolog 1 (RAF1) K07223 NM_002821 Homo sapiens PTK7 protein tyrosine kinase 7 (PTK7), transcript variant PTK7-1 K07225 NM_002758 Homo sapiens mitogen-activated protein kinase kinase 6 (MAP2K6) K07226 NM_002732
47、Homo sapiens protein kinase, cAMP-dependent, catalytic, gamma (PRKACG) K07229 NM_002741 Homo sapiens protein kinase N1 (PKN1), transcript variant 2 K07232 NM_002744 Homo sapiens protein kinase C, zeta (PRKCZ), transcript variant 1 K07233 NM_002748 Homo sapiens mitogen-activated protein kinase 6 (MAP
48、K6) K07234 NM_002751 Homo sapiens mitogen-activated protein kinase 11 (MAPK11) K07235 NM_002610 VARIANT 1 of Homo sapiens pyruvate dehydrogenase kinase, isozyme 1 (PDK1), nuclear gene encoding mitochondrial protein K07236 NM_002610 Homo sapiens pyruvate dehydrogenase kinase, isozyme 1 (PDK1), nuclea
49、r gene encoding mitochondrial protein K07237 NM_002611 Homo sapiens pyruvate dehydrogenase kinase, isozyme 2 (PDK2) K07239 NM_002613 VARIANT 1 of Homo sapiens 3-phosphoinositide dependent protein kinase-1 (PDPK1), K07240 NM_002613 Homo sapiens 3-phosphoinositide dependent protein kinase-1 (PDPK1), transcript variant 1 Gene TargetTMsiRNA Set 产品目录 Cat.No. ACCN No. Description K07241 NM_002376 VARIANT 1 of Homo sapiens MAP/microtubule affinity-regulating kinase 3 (MARK3) K07242 NM_002376 Homo sapiens MAP/microtubule a
