包含372個經典的、進化過程保守且具有顯著功能的mature miRNAs-康成生物丨數譜生物

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nrStar? Canonical Conserved miRNA PCR芯片

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Arraystar nrStar? Canonical Conserved miRNA PCR芯片(H)旨在通過qPCR技術檢測372個經典的、進化過程保守且具有顯著功能的mature miRNAs。此款PCR芯片能很好地消除miRNA前體、家族其他成員和isomiRs的干擾，同時還能夠準確地對低量樣本進行檢測，為科研人員提供一種準確、簡單和快捷的miRNA研究方案。

產品列表

產品名稱	貨號	規格	描述
nrStar? Human Canonical Conserved miRNA PCR Array	AS-NR-005	384 well / plate	包含372個經典的、進化過程保守且具有顯著功能的mature miRNAs
nrStar? Human Canonical Conserved (Roche Light Cycler 480)	AS-NR-005-R	384 well / plate	包含372個經典的、進化過程保守且具有顯著功能的mature miRNAs

優勢

? 專注于經典、進化保守且具有顯著功能的mature miRNAs

? 關注于具有更高表達水平的mature miRNAs而非star miRNAs

? 通過Two-tailed qPCR技術高效地消除前體、isomiRs及家族其他成員的干擾

? 詳細地注釋了miRNA啟動子、初級轉錄本及宿主基因等信息

? 方便快捷的預擴增步驟幫助準確地檢測低量樣本

為保證準確地檢測miRNA表達水平，消除前體、isomiRs及家族其他成員的影響，推薦使用rtStar? First-Strand cDNA Synthesis Kit (3’and 5’adaptor) (Cat# AS-FS-003)進行cDNA合成。針對低量樣本（100pg至200ng RNA），建議使用rtStar? PreAMP cDNA Synthesis Kit (Cat# AS-FS-006)進行cDNA合成及預擴增步驟。

嚴謹的目標miRNA篩選收錄流程

圖1.嚴謹地篩選經典、進化保守的miRNAs用于PCR芯片檢測。首先分析miRBase中所有pre-miRNA條目，挑選出經典pre-miRNA分子；其次將這些pre-miRNA進行系統發生學分析，找出進化過程中保守的miRNA；最后根據成熟miRNA的表達量，挑選出具有更高表達水平的mature miRNAs用于PCR芯片檢測。

專注于經典的miRNAs

miRBase收錄的miRNAs中，大約僅有1/3的人類miRNA基因、1/6的后生動物miRNA基因被認為是真實的miRNA基因（authentic miRNAs）[1]。經典miRNAs（Canonical miRNAs）擁有更為廣譜的表達，同時也能在不同樣本中具有差異性的表達（圖2）[2]。反之，大部分的非經典miRNAs（Noncanonical miRNAs）具有很差的保守性以及更低的表達水平，這預示著它們不具備顯著的基因表達調控能力[3]。故而，我們有理由在miRNA研究過程中關注于這些具有顯著功能的miRNA類群——經典miRNAs。

圖2. 經典miRNAs及mirtrons在不同樣本中的表達分布。

研究進化保守的miRNAs

與進化后期出現的miRNA基因相比，保守的miRNA（Conserved miRNAs）擁有更高的表達水平[3, 4]。在進化過程中新出現的miRNA基因往往對機體發揮中性亦或不利的調控功能，并且它們中的大部分在進化過程中慢慢地消失了[5]。然而保守的miRNAs的生物學功能則經歷了進化篩選，是生物體正常運轉所必需的。實際上保守的miRNAs能夠調控比非保守miRNAs更多的靶基因，并且這些靶基因中很大一部分行使轉錄因子功能（圖3）[6-8]。

圖3. 保守miRNAs（左）及非保守miRNAs（右）的靶基因功能統計分析。保守miRNAs主要調控轉錄因子的基因表達，而非保守miRNAs的靶基因功能則相對彌散。

聚焦于mature miRNAs而非star miRNAs

miRNA前體在Dicer酶的作用下產生雙鏈RNA，一條指導鏈和一條隨從鏈。當兩條鏈的表達水平相差2倍以上時，更高的成為mature miRNAs，而另一條則為star miRNAs；如若兩條鏈表達水平相當，則兩條鏈均稱為mature miRNAs[1]。再者，Star miRNAs發揮更為微弱的生物學功能。進化分析發現，star miRNAs擁有比mature miRNAs更高的堿基替代率，這種現象在seed區域顯得更為明顯（>46倍的差異）（圖4）。

圖4. Mature miRNAs和Star miRNAs的堿基替代率。n為所有堿基替代次數統計。

可區分前體、isomiRs及family members的two-tailed qPCR技術

由于miRNA有長度短小、isomiRs的存在、家族成員間高度同源以及特定樣品中低表達豐度等特點，使得miRNA的表達檢測顯得更為困難。雖然qPCR作為miRNA表達檢測中的“金標準”，然而傳統的qPCR方法也不能夠解決miRNA檢測中的困難。這是由于qPCR方法依賴于引物3’末端的差異，然而對于5’端的差異則愛莫能助[9, 10]。而傳統miRNA qPCR檢測方法僅僅依賴一條上游引物進行區分，下游多為通用引物。而生命體內有眾多RNAs與miRNAs在5’端存在單堿基差異，例如isomiRs以及同一家族其他miRNA成員。再者Seed序列位于5’區域，是miRNA識別靶基因的關鍵所在。Arraystar開發的two-tailed qPCR技術通過延伸miRNA兩端，使得區分前體、isomiRs以及家族其他成員更為容易。再者，該技術能夠方便快捷地進行低量樣本（低至100pg RNA）的預擴增操作（圖5）。

圖5. 利用Two-tailed qPCR技術及Poly-A qPCR技術分別對人工合成的miR-515家族miRNAs進行交叉檢測。

針對目標miRNAs進行全面注釋

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.

參考文獻：

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.

適用的qPCR儀：

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)

非常高度進化保守miRs (6):

miR-31-5p, miR-96-5p, miR-182-5p, miR-184, miR-210-3p, miR-375-3p

高度進化保守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

進化保守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

nrStar? Canonical Conserved miRNA PCR芯片

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