nrStar™ Canonical Conserved miRNA PCR芯片

• 描述
• 产品特点
• 实验流程
• 数据库

• Arraystar  nrStar™ Canonical Conserved miRNA PCR芯片(H)旨在通过qPCR技术检测372个经典的、进化过程保守且具有显著功能的mature miRNAs。此款PCR芯片能很好地消除miRNA前体、家族其他成员和isomiRs的干扰，同时还能够准确地对低量样本进行检测，为科研人员提供一种准确、简单和快捷的miRNA研究方案。

  产品列表

  产品名称	货号	规格	描述
  nrStar™ Human Canonical Conserved miRNA PCR Array	AS-NR-005	384 well / plate	包含372个经典的、进化过程保守且具有显著功能的mature miRNAs
  nrStar™ Human Canonical Conserved (Roche Light Cycler 480)	AS-NR-005-R	384 well / plate

  优势

  • 专注于经典、进化保守且具有显著功能的mature miRNAs

  • 关注于具有更高表达水平的mature miRNAs而非star miRNAs

  • 通过Two-tailed qPCR技术高效地消除前体、isomiRs及家族其他成员的干扰

  • 详细地注释了miRNA启动子、初级转录本及宿主基因等信息

  • 方便快捷的预扩增步骤帮助准确地检测低量样本

  为保证准确地检测miRNA表达水平，消除前体、isomiRs及家族其他成员的影响，推荐使用rtStar™ First-Strand cDNA Synthesis Kit (3’and 5’adaptor) (Cat# AS-FS-003)进行cDNA合成。针对低量样本（100pg至200ng RNA），建议使用rtStar™ PreAMP cDNA Synthesis Kit (Cat# AS-FS-006)进行cDNA合成及预扩增步骤。

• 严谨的目标miRNA筛选收录流程

  

  图1.严谨地筛选经典、进化保守的miRNAs用于PCR芯片检测。首先分析miRBase中所有pre-miRNA条目，挑选出经典pre-miRNA分子；其次将这些pre-miRNA进行系统发生学分析，找出进化过程中保守的miRNA；最后根据成熟miRNA的表达量，挑选出具有更高表达水平的mature miRNAs用于PCR芯片检测。

  专注于经典的miRNAs

  miRBase收录的miRNAs中，大约仅有1/3的人类miRNA基因、1/6的后生动物miRNA基因被认为是真实的miRNA基因（authentic miRNAs）[1]。经典miRNAs（Canonical miRNAs）拥有更为广谱的表达，同时也能在不同样本中具有差异性的表达（图2）[2]。反之，大部分的非经典miRNAs（Noncanonical miRNAs）具有很差的保守性以及更低的表达水平，这预示着它们不具备显著的基因表达调控能力[3]。故而，我们有理由在miRNA研究过程中关注于这些具有显著功能的miRNA类群——经典miRNAs。

  

  
  

  图2. 经典miRNAs及mirtrons在不同样本中的表达分布。

  研究进化保守的miRNAs

  与进化后期出现的miRNA基因相比，保守的miRNA（Conserved miRNAs）拥有更高的表达水平[3, 4]。在进化过程中新出现的miRNA基因往往对机体发挥中性亦或不利的调控功能，并且它们中的大部分在进化过程中慢慢地消失了[5]。然而保守的miRNAs的生物学功能则经历了进化筛选，是生物体正常运转所必需的。实际上保守的miRNAs能够调控比非保守miRNAs更多的靶基因，并且这些靶基因中很大一部分行使转录因子功能（图3）[6-8]。

  

  图3. 保守miRNAs（左）及非保守miRNAs（右）的靶基因功能统计分析。保守miRNAs主要调控转录因子的基因表达，而非保守miRNAs的靶基因功能则相对弥散。

  聚焦于mature miRNAs而非star miRNAs

  miRNA前体在Dicer酶的作用下产生双链RNA，一条指导链和一条随从链。当两条链的表达水平相差2倍以上时，更高的成为mature miRNAs，而另一条则为star miRNAs；如若两条链表达水平相当，则两条链均称为mature miRNAs[1]。再者，Star miRNAs发挥更为微弱的生物学功能。进化分析发现，star miRNAs拥有比mature miRNAs更高的碱基替代率，这种现象在seed区域显得更为明显（>46倍的差异）（图4）。

  

  图4. Mature miRNAs和Star miRNAs的碱基替代率。n为所有碱基替代次数统计。

  可区分前体、isomiRs及family members的two-tailed qPCR技术

  由于miRNA有长度短小、isomiRs的存在、家族成员间高度同源以及特定样品中低表达丰度等特点，使得miRNA的表达检测显得更为困难。虽然qPCR作为miRNA表达检测中的“金标准”，然而传统的qPCR方法也不能够解决miRNA检测中的困难。这是由于qPCR方法依赖于引物3’末端的差异，然而对于5’端的差异则爱莫能助[9, 10]。而传统miRNA qPCR检测方法仅仅依赖一条上游引物进行区分，下游多为通用引物。而生命体内有众多RNAs与miRNAs在5’端存在单碱基差异，例如isomiRs以及同一家族其他miRNA成员。再者Seed序列位于5’区域，是miRNA识别靶基因的关键所在。Arraystar开发的two-tailed qPCR技术通过延伸miRNA两端，使得区分前体、isomiRs以及家族其他成员更为容易。再者，该技术能够方便快捷地进行低量样本（低至100pg RNA）的预扩增操作（图5）。

  

  图5. 利用Two-tailed qPCR技术及Poly-A qPCR技术分别对人工合成的miR-515家族miRNAs进行交叉检测。

  针对目标miRNAs进行全面注释

  Annotation	Implications
  Tissue-specific	The miRNAs can have the tissue-specific functions in the corresponding tissue.
  Cell-specific	Cell-type specific miRNAs tend to target proteins at crucial bottleneck positions in the corresponding regulatory networks.
  Detection in Serum/plasma	miRNAs in serum/plasma having good potentials in biomarker applications.
  Promoter name	Information about the miRNA biogenesis. Also, many miRNA host genes are associated with the target gene activity either synergistically or antagonistically.
  Promoter Annotation
  Primary miRNA
  Host Gene
  Intronic/intergenic
  Seed family	miRNAs in the same seed family are evolutionarily closer and have at least partially redundant functions.
  Clustered miRNAs	miRNAs clustered in the genome generally have a similar expression level. They may regulate overlapping sets of target genes through convergent evolution.

  参考文献：

  1. Fromm B, Billipp T, Peck LE, Johansen M, Tarver JE, King BL, Newcomb JM, Sempere LF, Flatmark K, Hovig E et al: A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome. Annual review of genetics 2015, 49:213-242.

  2. Wen J, Ladewig E, Shenker S, Mohammed J, Lai EC: Analysis of Nearly One Thousand Mammalian Mirtrons Reveals Novel Features of Dicer Substrates. PLoS computational biology 2015, 11(9):e1004441.

  3. Bartel DP: Metazoan MicroRNAs. Cell 2018, 173(1):20-51.

  4. Nozawa M, Miura S, Nei M: Origins and evolution of microRNA genes in Drosophila species. Genome biology and evolution 2010, 2:180-189.

  5. Meunier J, Lemoine F, Soumillon M, Liechti A, Weier M, Guschanski K, Hu H, Khaitovich P, Kaessmann H: Birth and expression evolution of mammalian microRNA genes. Genome research 2013, 23(1):34-45.

  6. Agarwal V, Bell GW, Nam JW, Bartel DP: Predicting effective microRNA target sites in mammalian mRNAs. eLife 2015, 4.

  7. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB: Prediction of mammalian microRNA targets. Cell 2003, 115(7):787-798.

  8. Fahlgren N, Howell MD, Kasschau KD, Chapman EJ, Sullivan CM, Cumbie JS, Givan SA, Law TF, Grant SR, Dangl JL et al: High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PloS one 2007, 2(2):e219.

  9. Git A, Dvinge H, Salmon-Divon M, Osborne M, Kutter C, Hadfield J, Bertone P, Caldas C: Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression. Rna 2010, 16(5):991-1006.

  10. Lefever S, Pattyn F, Hellemans J, Vandesompele J: Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays. Clinical chemistry 2013, 59(10):1470-1480.

• 

  适用的qPCR仪：

  ABI ViiA™ 7

  ABI 7500 & ABI 7500 FAST

  ABI 7900HT

  ABI QuantStudio™ 6 Flex Real-Time PCR system

  ABI QuantStudio™ 7 Flex Real-Time PCR system

  ABI QuantStudio™ 12K Flex Real-Time PCR System

  Bio-Rad CFX384

  Bio-Rad iCycler & iQ Real-Time PCR Systems

  Eppendorf Realplex

  QIAGEN Rotor Gene Q 100

  Roche LightCycler 480

  Stratagene Mx3000

  Roche LightCycler 480

• Human Canonical Conserved miRNA列表 (372 miRNAs)

  非常高度进化保守miRs (6): miR-31-5p, miR-96-5p, miR-182-5p, miR-184, miR-210-3p, miR-375-3p 高度进化保守miRs (162): let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, let-7e-5p, let-7f-5p, let-7g-5p, let-7i-5p, miR-1-3p, miR-7-5p, miR-9-5p, miR-10a-5p, miR-10b-5p, miR-15a-5p, miR-15b-5p, miR-16-5p, miR-17-5p, miR-18a-5p, miR-18b-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-20b-5p, miR-21-5p, miR-22-3p, miR-23a-3p, miR-23b-3p, miR-24-3p, miR-25-3p, miR-26a-5p, miR-26b-5p, miR-27a-3p, miR-27b-3p, miR-29a-3p, miR-29b-3p, miR-29c-3p, miR-30a-5p, miR-30b-5p, miR-30c-5p, miR-30d-5p, miR-30e-5p, miR-32-5p, miR-33a-5p, miR-33b-5p, miR-34a-5p, miR-34b-5p, miR-34c-5p, miR-92a-3p, miR-92b-3p, miR-93-5p, miR-98-5p, miR-99a-5p, miR-99b-5p, miR-100-5p, miR-101-3p, miR-103a-3p, miR-106a-5p, miR-106b-3p, miR-106b-5p, miR-107, miR-122-5p, miR-125a-5p, miR-125b-5p, miR-126-3p, miR-126-5p, miR-128-3p, miR-129-5p, miR-130a-3p, miR-130b-3p, miR-132-3p, miR-133a-3p, miR-133b, miR-135a-5p, miR-135b-5p, miR-137-3p, miR-138-5p, miR-139-5p, miR-140-3p, miR-141-3p, miR-142-3p, miR-142-5p, miR-143-3p, miR-144-5p, miR-145-5p, miR-146a-5p, miR-146b-5p, miR-147b-3p, miR-148a-3p, miR-148b-3p, miR-150-5p, miR-152-3p, miR-153-3p, miR-155-5p, miR-181a-5p, miR-181b-5p, miR-181c-5p, miR-181d-5p, miR-183-5p, miR-187-3p, miR-190a-5p, miR-191-5p, miR-192-5p, miR-193a-5p, miR-193b-3p, miR-193b-5p, miR-194-5p, miR-195-5p, miR-196a-5p, miR-196b-5p, miR-199a/b-3p, miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-202-3p, miR-202-5p, miR-203a-3p, miR-204-5p, miR-205-5p, miR-206, miR-208a-3p, miR-208b-3p, miR-211-5p, miR-212-5p, miR-214-3p, miR-215-5p, miR-216b-5p, miR-217-5p, miR-218-5p, miR-219a-2-3p, miR-219a-5p, miR-221-3p, miR-222-3p, miR-223-3p, miR-301a-3p, miR-301b-3p, miR-302a-3p, miR-302b-3p, miR-302c-3p, miR-302d-3p, miR-338-3p, miR-363-3p, miR-365a-3p, miR-383-5p, miR-424-5p, miR-425-5p, miR-429, miR-449a, miR-449b-5p, miR-449c-5p, miR-451a, miR-454-3p, miR-455-3p, miR-455-5p, miR-489-3p, miR-490-3p, miR-497-5p, miR-499a-5p, miR-503-5p, miR-551a, miR-551b-3p, miR-802, miR-875-5p 进化保守miRs (204): miR-28-3p, miR-105-5p, miR-127-3p, miR-134-5p, miR-136-3p, miR-149-5p, miR-151a-3p, miR-151a-5p, miR-154-3p, miR-154-5p, miR-185-5p, miR-186-5p, miR-188-5p, miR-197-3p, miR-224-5p, miR-296-5p, miR-299-3p, miR-299-5p, miR-323a-3p, miR-323b-3p, miR-324-5p, miR-326, miR-329-3p, miR-330-3p, miR-331-3p, miR-335-5p, miR-337-3p, miR-339-3p, miR-339-5p, miR-340-5p, miR-342-3p, miR-345-5p, miR-346, miR-361-3p, miR-361-5p, miR-362-5p, miR-369-3p, miR-369-5p, miR-370-3p, miR-371a-5p, miR-372-3p, miR-373-3p, miR-374a-3p, miR-374a-5p, miR-374b-5p, miR-376a-3p, miR-376b-3p, miR-376c-3p, miR-377-3p, miR-377-5p, miR-378a-3p, miR-379-5p, miR-380-3p, miR-380-5p, miR-381-3p, miR-382-3p, miR-382-5p, miR-409-3p, miR-410-3p, miR-411-5p, miR-412-5p, miR-421, miR-423-3p, miR-423-5p, miR-431-5p, miR-432-5p, miR-433-3p, miR-448, miR-450a-5p, miR-450b-5p, miR-452-5p, miR-483-5p, miR-485-5p, miR-486-5p, miR-487a-5p, miR-487b-3p, miR-488-3p, miR-488-5p, miR-491-5p, miR-493-5p, miR-494-3p, miR-495-3p, miR-496, miR-498-5p, miR-500a-3p, miR-500b-5p, miR-501-3p, miR-502-3p, miR-504-5p, miR-506-3p, miR-506-5p, miR-507, miR-508-3p, miR-510-5p, miR-511-3p, miR-511-5p, miR-513b-5p, miR-514a-3p, miR-514b-5p, miR-516b-5p, miR-518b, miR-518c-3p, miR-518d-5p, miR-518f-3p, miR-519c-3p, miR-520a-3p, miR-520d-3p, miR-522-3p, miR-523-3p, miR-524-3p, miR-524-5p, miR-525-3p, miR-525-5p, miR-532-5p, miR-539-3p, miR-541-5p, miR-542-3p, miR-543, miR-545-5p, miR-574-3p, miR-576-5p, miR-577, miR-579-5p, miR-582-3p, miR-584-5p, miR-589-5p, miR-590-3p, miR-597-3p, miR-598-3p, miR-605-3p, miR-624-5p, miR-628-5p, miR-651-5p, miR-652-3p, miR-654-3p, miR-656-3p, miR-660-5p, miR-671-3p, miR-671-5p, miR-675-3p, miR-676-3p, miR-708-5p, miR-744-5p, miR-758-3p, miR-760, miR-769-5p, miR-770-5p, miR-873-3p, miR-873-5p, miR-874-3p, miR-876-3p, miR-885-5p, miR-887-3p, miR-887-5p, miR-888-5p, miR-889-3p, miR-890, miR-892a, miR-892b, miR-892c-3p, miR-934, miR-935, miR-944, miR-1180-3p, miR-1185-1-3p, miR-1185-2-3p, miR-1185-5p, miR-1197, miR-1247-5p, miR-1249-3p, miR-1251-5p, miR-1264, miR-1271-5p, miR-1277-5p, miR-1283, miR-1298-5p, miR-1301-3p, miR-1307-3p, miR-1307-5p, miR-1323, miR-1343-3p, miR-1911-5p, miR-1912-3p, miR-2114-3p, miR-2114-5p, miR-2355-3p, miR-2681-3p, miR-2681-5p, miR-3059-5p, miR-3085-3p, miR-3140-3p, miR-3145-3p, miR-3146, miR-3173-5p, miR-3200-3p, miR-3613-5p, miR-3617-5p, miR-3934-5p, miR-4524a-3p, miR-4677-3p, miR-4766-3p, miR-4766-5p, miR-6529-5p, miR-9851-3p