基本描述

别名	UltraBio™ Oligo (dT)25 磁珠 (mRNA 磁珠)
稳定性与储存	4℃保存，一年有效。
英文名称	UltraBio™ Oligo (dT)25 Magnetic Beads (mRNA磁珠)
储存温度	2-8°C储存
运输条件	冰袋运输
产品介绍	阿拉丁的UltraBio™ Oligo (dT)25 Magnetic Beads (mRNA磁珠)，也称mRNA Magnetic Beads或Oligo dT磁珠(Oligo dT Magnetic Beads)，是一种使用Oligo (dT)25包被的磁珠，可与mRNA等尾部的poly(A)互补配对，用于稳定、高效、便捷地直接从动物组织或培养的动物细胞中或从总RNA中快速分离纯化获得高纯度完整的带有poly(A)的mRNA等RNA的磁珠。本产品纯化的带有poly(A)的mRNA、lncRNA等，可直接应用于RT-PCR、qPCR、高通量测序、mRNA文库的构建、mRNA的m6', "A等修饰分析、固相cDNA文库构建、Northern blot分析、RACE等分子生物学实验，还可用于mRNA疫苗的研发，如mRNA疫苗的纯化、3' Poly A尾长度检测时Poly A尾短链的分离纯化等1-2]。本产品分离纯化的poly(A) RNA中也可包含带有poly(A)的lncRNA等，但通常主要为mRNA，因此也常被称为mRNA磁珠。一个典型的哺乳动物细胞中，四种主要的大分子的质量和占比为：RNA, ∼20pg (1%); DNA, ~7pg (0.3%); protein, ∼500pg (20%); polysaccharide (多糖), ∼2μg (78.7%)。信使RNA (messenger RNA, 简称mRNA) 约占总RNA质量的4%，核糖体RNA (ribosomal RNA, 简称rRNA)约占80% 3]。本产品的原理和主要操作流程如图1所示。UltraBio™ Oligo (dT)25磁珠表面共价修饰了25聚dT序列即Oligo (dT)25，当真核细胞、动植物组织的裂解液或抽提的总RNA与UltraBio™ Oligo (dT)25', "磁珠混合后，磁珠表面的寡聚dT序列与mRNA等3'端的poly(A)进行碱基配对而特异性结合，然后在外界磁场的作用下，磁珠与相应溶液可以快速而高效地分离，经洗涤充分去除杂质，最后用洗脱液将mRNA等从磁珠上洗脱下来，即可获得高纯度完整mRNA等带有poly(A)的RNA4-5]。图1.UltraBio™ Oligo (dT)25 Magnetic Beads (mRNA磁珠) 用于mRNA抽提的工作原理示意图。本产品盒具有提取效果稳定、纯度高、速度快、操作便捷等优点。本产品的mRNA提取体系经过反复测试和优化，能分离纯化获得总RNA中90%以上的mRNA，能从106个细胞中抽提约0.1-1µg mRNA，20mg小鼠肝组织能抽提约0.2-2µg mRNA，20mg小鼠肺组织能抽提约0.1-1µg mRNA。仅需裂解、结合、洗涤、洗脱等简单的操作，整个纯化过程不超过15分钟即可完成。所有操作都在同一个离心管中完成，操作便捷。提取的total RNA使用本产品进行mRNA的纯化效果参考图2。图2.UltraBio™ Oligo (dT)25 Magnetic Beads (mRNA磁珠) 用于从总RNA中纯化mRNA的效果图。使用RNAeasy™动物RNA抽提试剂盒(离心柱式) 抽提293T细胞的总RNA，同时使用本产品进行mRNA的纯化，然后使用UltraBio™ SYBR Green One-Step qRT-PCR Kit 进行qRT-PCR检测，使用Actin、B2M和GAPDH的三对内参引物对比总RNA (Total RNA)和纯化的mRNA的Ct值。图中可见mRNA纯化前后的Ct值基本一致，说明mRNA的纯化效果接近100%。实际结果会因实验条件、检测仪器等的不同而存在差异，图中数据仅供参考。本产品磁珠粒径约为200nm，浓度约为5mg/ml。每毫克磁珠偶联的Oligo (dT)25约为300-400pmol，每毫克磁珠可纯化约2-3µg mRNA等poly(A) RNA，即每毫升磁珠可以纯化约10-15µg mRNA等poly(A) RNA。对于常规的mRNA纯化，按照每个样品使用20μl磁珠悬浊液，每1ml本磁珠可用于50次mRNA纯化。4℃保存，一年有效。其中UltraBio™ Oligo (dT)25磁珠长期不使用时，可以使用说明： 1.准备工作。 a.缓冲液的准备。根据实验需要配制相关缓冲液，参考配方如下配制： Buffers Recipe Binding Buffer 20mM Tris-HCl (pH7.5), 1M LiCl, 2mM EDTA Lysis Buffer 100mM Tris-HCl (pH7.5), 500mM LiCl, 10mM EDTA,1% LiDS, 5mM DTT Washing Buffer I 10mM Tris-HCl (pH7.5), 150mM LiCl, 1mM EDTA, 0.1% LiDS Washing Buffer II 10mM Tris-HCl (pH7.5), 150mM LiCl, 1mM EDTA Elution Buffer 10mM Tris-HCl (pH7.5) 注：试剂和耗材的RNase-free要求参见注意事项；试剂在使用时平衡至室温，如有沉淀，可37℃预热10分钟。 b.细胞样品的准备。 (a)贴壁细胞用PBS洗涤一次；悬浮细胞1000-2000×g室温离心5分钟，弃上清，用PBS洗涤一次。 (b)按照细胞量加入推荐的裂解液。通过移液器吹打多次，裂解细胞至溶液变粘稠。 (c)选做：加入溶液裂解液后样品可能会比较粘稠，可适当超声或使用1ml注射器反复抽吸打断基因组DNA从而使粘稠感消失。此操作会产生泡沫，但不影响mRNA的得率。 (d)4℃以14,000×g离心5分钟，并将上清转移至一个新的离心管。上清可用于mRNA纯化，或保存在-80℃备用。 c.动物组织样品的准备。 (a)取所需量的动物组织，置于液氮中研磨成粉末，立即加入推荐量的裂解液。也可将组织置于1.5ml离心管中，迅速加入推荐量的冰浴预冷的裂解液，用微型电动匀浆器匀浆，或者用普通玻璃匀浆器进行匀浆。 (b)4℃以14,000×g离心5分钟，并将上清转移至一个新的离心管。上清可用于mRNA纯化，或保存在-80℃备用。 d. Oligo (dT)25磁珠的准备。 (a)将磁珠溶液从4℃冰箱取出，适当涡旋震荡或反复颠倒以确保磁珠充分混匀。参考下表，根据总RNA量和总细胞量或组织重量，取适量的 Oligo (dT)25磁珠悬液至一洁净离心管中。推荐使用 1.5毫升离心管(无色, Nuclease free) 。 Total RNA Beads required Washing Buffer II 1~10µg 20µl 200µl each time 10~30µg 40µl 200µl each time 30-50µg 100µl 200µl each time Cell number Tissue mass Lysis Buffer Beads Washing Buffer I Washing Buffer Ⅱ ＜5×105 ＜5mg 100µl 20µl 500µl 500µl each time 1~2×106 5~20mg 300µl 40µl 500µl 500µl each time 3~5×106 20~50mg 600µl 100µl 500µl 500µl each time (b)无论磁珠用量多少，按照每个样品200µl结合液的量，加入适量结合液洗涤磁珠，用移液器轻轻吹打重悬磁珠。置于磁力架上分离30秒，去除上清。重复本步骤1次。说明：如果样品数量超过5个，可以考虑将适量磁珠悬液先直接置于磁力架上分离30秒，去除上清，然后根据样品数量再加入适量结合液洗涤2次。 (c)按照每个样品100µl结合液的量，加入适量溶液结合液重悬磁珠。 2.从细胞或组织样品中分离纯化mRNA。 a.取上述制备好的细胞或组织样品与100µl洗涤后重悬的磁珠在室温下旋转混合5分钟。 b.置于磁力架上分离1分钟，去除上清。 c.室温下用500µl洗涤液I洗涤磁珠，磁分离30秒，去上清。 d.室温下用500µl洗涤液Ⅱ洗涤磁珠，磁分离30秒，去上清。重复本步骤1次。 e.根据后续实验需求，进行mRNA的洗脱： (a)从磁珠上洗脱mRNA：加入10-20µl洗脱液或Nuclease-free的水，如DEPC水或 Ultrapure Water (DNase/RNase-Free, Sterile) ，75-80℃孵育2分钟，磁分离30秒，然后将上清转移到新的Nulcease-free的离心管中，置于冰上待用。注：建议转移上清时保留少量液体以免吸到磁珠影响后续实验。纯化获得的mRNA极易降解，建议尽快进行后续实验。短时间内不使用，请置于-80℃保存。 (b)如果mRNA不洗脱直接用于后续实验，如固相cDNA文库构建等，用500µl洗涤液Ⅱ洗涤一次，再用后续实验中相应的缓冲液再洗涤一次，即可用于后续实验。 3.从Total RNA中纯化mRNA (以Total RNA的量为20µg为例)。 a.取100µl含有20µg Total RNA的样品与100µl结合液混合。注：如果20µg Total RNA不足100µl，可以用DEPC水或其它适当Nuclease-free溶液补足至100µl。 b.65ºC孵育2分钟以打开RNA的二级结构，孵育结束后迅速置于冰上。 c.将该200µl混合液与100µl洗涤后重悬的磁珠在室温下旋转混合5分钟。 d.置于磁力架上分离1分钟，去除上清。 e.室温下用200µl 洗涤液Ⅱ 洗涤磁珠，磁分离30秒，去上清。重复本步骤1次。 f.根据后续实验需求，进行mRNA的洗脱： (a)从磁珠上洗脱mRNA：加入10-20µl洗脱液或Nuclease-free的水，如DEPC水或 Ultrapure Water (DNase/RNase-Free, Sterile) ，75-80℃孵育2分钟，磁分离30秒，然后将上清转移到新的Nulcease-free的离心管中，置于冰上待用。注：建议转移上清时保留少量液体以免吸到磁珠影响后续实验。纯化获得的mRNA极易降解，建议尽快进行后续实验。短时间内不使用，请置于-80℃保存。 (b)如果mRNA不洗脱直接用于后续实验，如固相cDNA文库构建等，用后续实验中相应的缓冲液再洗涤一次，即可用于后续实验。参考文献： 1.Wommer L, Soerjawinata W, Ulber R, Kampeis P. Eng Life Sci. 2021. 21(10): 558–572. 2.Chaudhary N, Weissman D & Whitehead K A. Nat Rev Drug Discov. 2021.20, 817–838. 3.Wu J, Xiao J, Zhang Z, Wang X, Hu S, Yu J. Genomics Proteom Bioinforma. 2014. 12(2):57-63. 4.Michael R Green, Joseph Sambrook. Cold Spring Harb Protoc. 2019.10:711-714. 5.Nicholas M. Adams, Hali Bordelon, et al. ACS Applied Materials & Interfaces. 2015. 7(11):6062-6069. Aladdin's UltraBio™ Oligo (dT)25 Magnetic Beads, also known as mRNA Magnetic Beads or Oligo dT Magnetic Beads, allow for efficient, rapid, and convenient isolation of highly purified intact RNAs with poly(A) sequence such as mRNA from eukaryotic total RNA or from crude extracts of animal cells or tissues directly, based on base-pairing between the poly(A) tail of mRNA and the Oligo (dT)25 bound to the surface of the beads. The RNA such as mRNA and lncRNA with poly(A) purified by this product can be directly used in molecular biology applications such as RT-PCR, qPCR, RNA sequencing, library construction, RNA modification analysis, solid-phase cDNA library construction, Northern blot, and RACE, etc. It can also be used to develop mRNA vaccines, such as the purification of mRNA vaccines, the isolation of short strands with poly(A) tails for 3' Poly(A) tail-length assay, etc. [1-2].The mass and percentage weight of four major macromolecules in a typical mammalian cell are as follows Precautions： The whole process should be strictly free of RNase and DNase. For the removal of RNase from working environments, we recommend using RNase and DNase Away .All reagents and consumables used for this product should be RNase- and DNase-free, and should be handled with care to avoid contamination. RNase can be removed from contaminated consumables by soaking them in 0.01% DEPC water overnight, followed by autoclaving which can inactivate DNase in the mean while.Magnetic stands are required but not supplied in this product. We recommend using Mag™ Magnetic Separation Racks (FMS004, FMS008, FMS012, FMS016 or FMS024).When dispensing or using the magnetic beads, please vortex properly or invert the tube several times to fully resuspend the magnetic beads.Please use appropriate amount of sample as recommended in this manual and do not overload the beads. Otherwise, the beads may aggregate, which will affect the washing efficiency and thus the purity of isolated mRNA. If bead aggregation occurs, disperse the beads as much as possible during washing to ensure the purity of isolated RNA.Since mRNA is easily degraded, the isolated mRNA should be used for downstream applications such as RT-PCR as soon as possible. Store mRNA at -80 ℃ to minimize degradation.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Instructions for Use： 1. Preparation of reagents and samples.a. Prepare the buffers as follow. Equilibrate them to room temperature before use, and incubate at 37℃ for 10 minutes if there are any precipitates.Buffers Recipe Binding Buffer 20mM Tris-HCl (pH7.5), 1M LiCl, 2mM EDTA Lysis Buffer 100mM Tris-HCl (pH7.5), 500mM LiCl, 10mM EDTA,1% LiDS, 5mM DTT Washing Buffer I 10mM Tris-HCl (pH7.5), 150mM LiCl, 1mM EDTA, 0.1% LiDS Washing Buffer II 10mM Tris-HCl (pH7.5), 150mM LiCl, 1mM EDTA Elution Buffer 10mM Tris-HCl (pH7.5) b. Preparation of lysate from cell samples.(a) For adherent cells, wash once with PBS; for suspension cells, centrifuge at 1000-2000×g for 5 min at room temperature, discard the supernatant, and wash once with PBS.(b) By referring to the table below, add the recommended amount of Lysis Buffer for a specific number of cells. Mix well by pipetting and lyse cells until the solution becomes viscous.(c) (Optional) If sample lysate is too viscous, sonicate or repeatedly aspirate it using a 1 ml syringe to shear genomic DNA. This operation will produce foam, but does not affect the mRNA yield.(d) Centrifuge at 14,000×g for 5 min at 4℃ and transfer the supernatant to a new centrifuge tube. The supernatant is now ready for mRNA isolation or can be stored at -80℃ for later use.c. Preparation of lysate from animal tissues.(a) Take an appropriate amount of animal tissues as required, freeze in liquid nitrogen and grind into powder. Immediately add the recommended amount of Lysis Buffer by referring to the table below and homogenize. Alternatively, the tissue can be placed in a 1.5 ml centrifuge tube, add the recommended amount of ice-cold Lysis Buffer, and homogenize with a microelectric homogenizer, or with a regular glass homogenizer.(b) Centrifuge at 14,000×g for 5 min at 4℃ and transfer the supernatant to a new centrifuge tube. The supernatant is now ready for mRNA isolation or can be stored at -80℃ for later use. d. Preparation of Oligo (dT)25 Magnetic Beads.(a) Take out the Magnetic Beads from the 4℃ refrigerator and vortex or invert the tube several times to fully resuspend the beads. Depending on the total RNA amount, total number of cells or tissue weight, add an appropriate amount of Oligo (dT)25 bead suspension to a clean centrifuge tube, by referring to the table below. We recommend using 1.5ml Centrifuge Tubes (colorless, Nuclease free) ( Ultrapure Water . Incubate at 75-80℃ for 2min. Place the tube on a magnetic stand for 30 seconds, then transfer the supernatant to a new nuclease-free tube. The isolated RNA can be kept on ice for immediate use or stored at -80℃ for later use.Note: Do not take all of the liquid when transferring the supernatant to avoid absorbing the magnetic beads which will affect downstream applications. (b) If the bead-bound isolated mRNA is to be used in downstream applications, such as solid-phase cDNA library preparation, wash the beads one extra time with 500µl of Wash Buffer II, followed by one wash with the buffer used in the downstream application.3. Isolation of mRNA from total RNA.The following protocol is provided for mRNA purification from 20µg of total RNA starting material.a. Adjust the volume of the total RNA sample (20µg) to 100µl with nuclease-free water, and mix with 100µl of Binding Buffer.b. Incubate at 65℃ for 2 minutes to disrupt secondary structures of RNA. Immediately place on ice.c. Add the 200µl of total RNA to the 100µl of washed beads, mix well, and allow binding by rotating on a mixer for 5 minutes at room temperature.d. Place the tube on a magnetic stand for 1min and remove the supernatant carefully. e. Wash the beads with 200µl of Wash Buffer Ⅱ at room temperature, place the tube on magnetic stand for 30sec, and remove the supernatant. Repeat this step once. f. Elution of mRNA with one of the following methods based on the requirement of downstream applications. (a) Elution of mRNA from magnetic beads: After aspiration of Wash Buffer II, add 10-20µl of Elution Buffer or nuclease-free water, such as Ultrapure Water . Incubate at 75-80℃ for 2min. Place the tube on a magnetic stand for 30 seconds, then transfer the supernatant to a new nuclease-free tube. The isolated RNA can be kept on ice for immediate use or stored at -80℃ for later use.Note: Do not take all of the liquid when transferring the supernatant to avoid absorbing the magnetic beads and affecting downstream experiments.(b) If the bead-bound isolated mRNA is to be used in downstream applications, such as solid-phase cDNA library preparation, wash the beads once with the buffer used in the downstream application.

安全和危险性（GHS）

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质量标准

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溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

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分子量

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稀释计算器

计算配制储备溶液所需的稀释度。

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浓度 2 (C2)

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复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

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质量（小瓶）

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所需的复溶浓度

货号 (SKU)	包装规格	是否现货
O751564-1ml	1ml	期货
O751564-5ml	5ml	期货
O751564-20ml	20ml	期货
O751564-100ml	100ml	期货

UltraBio™ Oligo (dT)25 Magnetic Beads (mRNA磁珠)

库存信息

库存信息

库存信息

库存信息

基本描述

安全和危险性（GHS）

质量标准

质检证书（CoA,COO,BSE/TSE 和分析图谱)

溶液计算器

摩尔浓度计算器

稀释计算器

复溶计算器