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
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human canonical conserved mirna列表 (372 mirnas) |
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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 |
