计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,避光,室温。低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
miRNA提取试剂盒专用于从各种动物组织、植物组织、细胞、血清、血浆等样本中分离纯化miRNA，还可以提取siRNA，snRNA等其他小于200 nt的小分子RNA，同时也可用于总RNA的提取。本品将酚/胍裂解技术和硅基质膜纯化技术相结合，独特的裂解液在有效抑制RNases的同时，可以通过有机抽提的方法除去细胞或组织样品中的大部分DNA和蛋白。对于一些敏感的下游实验中，如需富集miRNA可适用该试剂盒单独对miRNA进行富集。本品适用样本范围广，制备的RNA纯度高，可直接用于敏感的下游应用，如Northern Blot分析，Real-Time PCR，Microarray Analysis等。
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自备试剂：
氯仿、无水乙醇（新开封或提取RNA专用）。
实验前准备及重要注意事项：
1．预防RNase污染，应注意以下几方面：
1）使用无RNase的塑料制品和枪头，避免交叉污染。
2）玻璃器皿应在使用前于180℃高温下干烤4小时，塑料器皿可在0.5 M NaOH中浸 泡10分钟，用水彻底冲洗后高压灭菌。
3）配制溶液应使用无RNase的水。
4）操作人员戴一次性口罩和手套，实验过程中要勤换手套。
2．提取的样品避免反复冻融，否则影响miRNA提取的量和质量。
3．第一次使用前应按照试剂瓶标签说明在Buffer RWT和Buffer RW2中加入无水乙醇。
4．所有离心步骤若无特殊说明均在室温下进行，且所有操作步骤动作要迅速。
操作步骤：
Protocol A：miRNA富集 （可直接用于敏感的下游实验）
1．样品处理
1a. 组织：将组织在液氮中磨碎。每30-50 mg组织加1 ml TRIzon Reagent，震荡混匀。样 品体积不超过TRIzon Reagent体积的十分之一。
1b. 单层培养细胞：吸去培养液，加入TRIzon Reagent，每10 cm2 加入1 ml TRIzon Reagent （裂解液用量视培养瓶面积而定）。
1c. 细胞悬液：离心得到细胞沉淀，弃上清。每5×106 -1×107 细胞加入1 ml TRIzon Reagent （细胞不需洗涤）。
1d. 血浆或血清：取200 μl血浆或血清样本，加入5倍体积TRIzon Reagent，震荡混匀30秒。
2．样品中加入TRIzon Reagent后反复吹打几次，使其充分裂解。室温放置5分钟，使 蛋白核酸复合物完全分离。
3．可选步骤：4℃ 12,000 rpm（~13,400×g）离心5分钟，取上清，转入一个新的离心 管（自备）中（如样品中含较多蛋白、脂肪、多糖等，可选做此步骤）。
4．向上清中加入氯仿，每使用1 ml TRIzon Reagent加入200 μl氯仿，盖好管盖，剧烈 振荡15秒，室温放置5分钟。
5．4℃ 12,000 rpm离心15分钟，样品分为三层：红色有机相，中间层，无色水相，将 上层无色水相移到一个新的离心管（自备）中。
6．向步骤5得到的溶液中加入1/3倍体积的无水乙醇， 混匀，将得到的溶液和沉淀一起 转入已装入收集管的吸附柱RM（Spin Columns RM）中。若一次不能将全部溶液加 入吸附柱，请分多次转入。12,000 rpm离心30秒，离心后弃掉吸附柱RM，保留流 出液。
7．向步骤6得到的溶液中加入2/3倍体积的无水乙醇，混匀。
8．将上步所得溶液和沉淀一起转入已装入收集管的吸附柱RS（Spin Columns RS）。 若一次不能将全部溶液加入吸附柱，请分多次转入。12,000 rpm离心30秒，倒掉收 集管中废液，将吸附柱RS重新放回收集管中。
9．向吸附柱RS中加入700 μl Buffer RWT（使用前检查是否加入无水乙醇），12,000 rpm离心 30秒, 倒掉收集管中的废液，将吸附柱RS重新放回收集管中。
10.向吸附柱RS中加入500 μl Buffer RW2（使用前检查是否加入无水乙醇）, 12,000 rpm离心 30秒，倒掉收集管中的废液，将吸附柱RS重新放回收集管中。
11.重复步骤10。
12.12,000 rpm离心1分钟，倒掉收集管中的废液。将吸附柱RS置于室温数分钟，以彻 底晾干。 注意：此步骤的目的是将吸附柱RS中残留的乙醇去除，乙醇的残留会影响后续的酶促反应（酶切、 PCR等）。
13.将吸附柱RS置于一个新的无RNase离心管中，向吸附柱中间部位加入30-50 μl RNase-Free Water，室温放置1分钟，12,000 rpm离心1分钟，收集RNA溶液，得 到的RNA溶液保存在-70℃，防止降解。
注意：
1）RNase-Free Water体积不应小于30 μl，体积过小影响回收率。
2）如果要提高RNA的产量，可用30-50 μl新的RNase-Free Water重复步骤13。
3）如果要提高RNA浓度，可将得到的溶液重新加入到吸附柱RS中，重复步骤13
Protocol B：总RNA的提取（提取的总RNA包括miRNA等其他<200 nt的小分子RNA） 1~5步骤同protocol A。
6．向步骤5得到的溶液中加入1.25倍体积的无水乙醇，混匀。
7．将上步所得到的溶液和沉淀一起转入已装入收集管的吸附柱（Spin Columns RM）中。 若一次不能将全部溶液加入吸附柱RM中，请分多次转入。12,000 rpm离心30秒，倒掉 收集管中的废液，将吸附柱RM重新放回收集管中。
8．向吸附柱RM中加入700 μl Buffer RWT（使用前检查是否加入无水乙醇）, 12,000 rpm离心30秒，倒掉收集管中的废液，将吸附柱RM重新放回收集管中。
9．向吸附柱RM中加入500 μl Buffer RW2（使用前检查是否加入无水乙醇）, 12,000 rpm离心30秒，倒掉收集管中的废液，将吸附柱RM重新放回收集管中。
10.重复步骤9。
11.12,000 rpm离心1分钟，倒掉收集管中的废液。将吸附柱RM置于室温数分钟，以彻 底晾干。
注意：此步骤的目的是将吸附柱RM中残留的乙醇去除，乙醇的残留会影响后续的酶促反应（酶切、 PCR等）。
12.将吸附柱RM转入新的无RNase离心管中，向吸附柱中间部位加入30-50 μl RNase-Free Water，室温放置1分钟，室温 12,000 rpm离心1分钟，收集RNA溶液，得到的RNA溶液 保存在-70℃，防止降解。
注意：
1）RNase-Free Water体积不应小于30 μl，体积过小影响回收率。
2）如果要提高RNA的产量，可用30-50 μl新的RNase-Free Water重复步骤12。
3）如果要提高RNA浓度，可将得到的溶液重新加入到吸附柱RM中，重复步骤12。
The miRNA extraction kit is specifically designed to isolate and purify miRNAs from various animal tissues, plant tissues, cells, serum, plasma and other samples. It can also extract small molecule RNAs such as siRNA and snRNA that are less than 200 nt, and can also be used for the extraction of total RNA. This product combines phenol/guanidine lysis technology and silicon matrix membrane purification technology. The unique lysis solution can effectively inhibit RNases while removing most of DNA and proteins from cell or tissue samples through organic extraction. For some sensitive downstream experiments, if miRNA enrichment is required, this kit can be used to enrich miRNA separately. This product is suitable for a wide range of samples, with high purity of prepared RNA, and can be directly used for sensitive downstream applications, such as Northern Blot analysis, Real Time PCR, Microarray Analysis, etc.
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Self prepared reagents:
chloroform, anhydrous ethanol (newly opened or dedicated for RNA extraction).
Preparation and important precautions before the experiment:
To prevent RNase pollution, attention should be paid to the following aspects:
1) Use RNase free plastic products and gun heads to avoid cross contamination.
2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.
3) Prepare the solution using water without RNase.
4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.
2. The extracted samples should avoid repeated freeze-thaw cycles, otherwise it will affect the quantity and quality of miRNA extraction.
Before the first use, anhydrous ethanol should be added to Buffer RWT and Buffer RW2 according to the instructions on the reagent bottle label.
4. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.
Operation steps:
Protocol A: miRNA enrichment (can be directly used for sensitive downstream experiments)
1. Sample processing
1a Organization: Grind the organization in liquid nitrogen. Add 1 ml of TRIzon Reagent to every 30-50 mg of tissue, shake and mix well. The sample volume shall not exceed one tenth of the volume of TRIzon Reagent.
1b Single layer culture of cells: Remove the culture medium, add TRIzon Reagent, and add 1 ml of TRIzon Reagent every 10 cm2 (the amount of lysis solution depends on the area of the culture bottle).
1c Cell suspension: Centrifuge to obtain cell precipitate, discard supernatant. Add 1 ml of TRIzon Reagent to every 5 x 106-1 x 107 cells (cells do not require washing).
1d Plasma or serum: Take 200 μ Add 5 times the volume of TRIzon Reagent to plasma or serum samples, shake and mix well for 30 seconds.
2. After adding TRIzon Reagent to the sample, blow it repeatedly several times to fully crack it. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.
3. Optional steps: Centrifuge at 4 ℃ 12000 rpm (~13400 × g) for 5 minutes, take the supernatant, and transfer it to a new centrifuge tube (provided by oneself) (if the sample contains more proteins, fats, polysaccharides, etc., this step can be performed).
4. Add chloroform to the supernatant and add 200 to every 1 ml of TRIzon Reagent used μ Chloroform, cover the tube, vigorously shake for 15 seconds, and let it sit at room temperature for 5 minutes.
Centrifuge at 5.4 ℃ and 12000 rpm for 15 minutes. The sample is divided into three layers: red organic phase, middle layer, and colorless aqueous phase. Transfer the upper colorless aqueous phase to a new centrifuge tube (self prepared).
6. Add 1/3 volume of anhydrous ethanol to the solution obtained in step 5, mix well, and transfer the obtained solution and precipitate together into the adsorption column RM (Spin Columns RM) that has been loaded into the collection tube. If you cannot add all the solution to the adsorption column at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the adsorption column RM after centrifugation, and retain the effluent.
7. Add 2/3 times the volume of anhydrous ethanol to the solution obtained in step 6 and mix well.
8. Transfer the solution and precipitate obtained from the previous step into the adsorption column RS (Spin Columns RS) that has been loaded into the collection tube. If you cannot add all the solution to the adsorption column at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.
9. Add 700 to the adsorption column RS μ L Buffer RWT (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.
10. Add 500 to the adsorption column RS μ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.
11. Repeat step 10.
12. Centrifuge at 12000 rpm for 1 minute and discard the waste liquid from the collection tube. Place the adsorption column RS at room temperature for a few minutes to thoroughly air dry. Note: The purpose of this step is to remove residual ethanol from the adsorption column RS, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
13. Place the adsorption column RS in a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column μ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store the obtained RNA solution at -70 ℃ to prevent degradation.
Attention:
1) The volume of RNase Free Water should not be less than 30 μ l. Small volume affects the recovery rate.
2) If you want to increase RNA production, you can use 30-50 μ Repeat step 13 for the new RNase Free Water.
3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column RS and repeat step 13
Protocol B: Extraction of total RNA (including miRNA and other small molecule RNAs<200 nt), steps 1-5 are the same as protocol A.
6. Add 1.25 times the volume of anhydrous ethanol to the solution obtained in step 5 and mix well.
7. Transfer the solution and precipitate obtained from the previous step into the spin columns RM that have been loaded into the collection tube. If you cannot add all the solution to the adsorption column RM at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.
8. Add 700 to the adsorption column RM μ L Buffer RWT (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.
9. Add 500 to the adsorption column RM μ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.
10. Repeat step 9.
11. Centrifuge at 12000 rpm for 1 minute and discard the waste liquid from the collection tube. Place the adsorption column RM at room temperature for a few minutes to thoroughly air dry.
Attention:
The purpose of this step is to remove residual ethanol from the adsorption column RM, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
12. Transfer the adsorption column RM into a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column μ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store the obtained RNA solution at -70 ℃ to prevent degradation.
Attention:
1) The volume of RNase Free Water should not be less than 30 μ l. Small volume affects the recovery rate.
2) If you want to increase RNA production, you can use 30-50 μ Repeat step 12 for the new RNase Free Water.
3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column RM and repeat step 12.
规格
英文名称
miRNA Purification Kit
储存与运输
储存条件
2-8°C储存,避光,室温
运输条件
低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
溶液计算器
常见问题
本产品应如何储存？
请于2–8 °C、避光条件下保存。本品对光敏感，请保存在原始的避光或棕色容器中。
本产品如何运输？
本产品采用湿冰冷藏运输。收货后请立即拆包，并按上述储存条件保存。
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每个订单都会随附分析证书（COA），您也可以从账户订单历史中下载。如需订购前的COA，请联系您的客户代表，或使用PDP上的"申请COA"按钮——我们将提供代表性批次的COA。
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