计算溶液所需的质量、体积或浓度。
微量样本总RNA提取试剂盒 (DNase I)
有货
为什么选择此级别
BioReagent,分子生物学级 ，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,室温,-20°C储存。超低温运输 。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
本产品采用全新技术从微量的组织和细胞中（少至10个细胞）进行RNA提取，本产品在裂解液中添加了特殊组分，可大幅度提高RNA和硅基质膜的选择性结合能力。同时，在RNA提取过程中，还加入DNase I，可完全去除痕量DNA杂质，而DNase I和其他残留的物质会在随后的漂洗过程中去除，从而有效地保证了提取RNA的得率和纯度。
与其他提取RNA的普通方法相比，微量样品RNA提取试剂盒将所有<200 bp的RNA（5.8S rRNA，5SrRNA和tRNAs等）选择性地筛除，而所有>200 bp的RNA则被富集、分离和纯化。在本说明书中，对于不同的样品有不同的操作步骤，这些步骤的区别主要在于样品的裂解和结合至硅基质膜的条件不同，当RNA结合至硅基质膜后，后续步骤基本类似。
注意事项
1. 自备试剂与耗材
β-巯基乙醇，无水乙醇，1.5 mL RNase-Free离心管
2. 样品使用量的确定
为了使用本试剂盒得到高产量和高纯度的RNA，使用样品的量是否得当是很重要的，因为吸附柱的吸附量以及裂解液的用量有一定限制，只有保证样品充分的裂解及RNA充分吸附在硅基质膜上，RNA的得率才有保证。
表1是该试剂盒的技术指标，表2是在不同培养条件下细胞的数量。
表1. 微量样品RNA提取试剂盒技术指标
| 吸附柱最大容量 | 700 µL |
| 提取RNA的长度 | >200 bp |
| 最小洗脱体积 | 10 µL |
| 样品使用最大量（动物细胞） | 5× 10⁵ |
| 样品使用最大量（动物组织） | 5 mg |
注意：如果超出该试剂盒的最大吸附能力，RNA的产量可能会降低，而如果样品未能裂解完全，RNA的产量也会降低。
表2. 不同培养条件下HeLa细胞的数量
| 细胞培养器皿 | 生长面积（cm²） | 细胞数量 |
| 96孔细胞培养板 | 0.32-0.6 | 4-5 × 10⁴ |
| 48孔细胞培养板 | 1 | 1× 10⁵ |
| 24孔细胞培养板 | 2 | 2.5× 10⁵ |
| 12孔细胞培养板 | 4 | 5× 10⁵ |
| 6孔细胞培养板 | 9.5 | 1× 10⁶ |
| 平皿 (35 mm) | 8 | 1× 10⁶ |
| 摇瓶 (40-50 mL) | 25 | 3× 10⁶ |
3. 样品的储存和处理
组织若不经过处理，其中的RNA处于无保护状态，容易被降解，所以新鲜组织应及时放入液氮中速冻并立即储存于-70℃，冻存的组织不能反复冻融以避免RNA的降解，样品也可以在匀浆后加入Buffer URL，储存于-70℃，冻存的样品可稳定储存数月。
操作前的注意事项
1. 为了防止RNA的降解，请避免未处理的样品在室温长时间放置，在组织固定前，RNA处于无保护状态，所以请将组织在液氮中速冻。
2. 保存在Buffer URL的组织可在-70℃储存数月。处理冻存的裂解液时，可在室温或37℃水浴中将样品完全解冻，而且请注意裂解液中的盐需要完全溶解，之后立即进行后续操作。（长时间37℃处理将会导致RNA的化学降解）。
3. 在使用前，请用RNase-Free ddH₂O配制70%乙醇溶液。
4. DNase I 储存液的配制：用1 mL注射器将550 μL RNase-Free ddH₂O注入装有DNase I 干粉的瓶中，轻柔混匀至溶解，分装后-30~-15℃储存（可保存9个月），每次根据用量需求取出。配制的DNase I 工作液置于2-8℃储存（可保存42天），避免反复冻融。
操作步骤
使用前准备：在12 mL Buffer RW2 中加入48 mL的无水乙醇。
一、从福尔马林固定的微切割样品中提取总RNA
在从福尔马林固定的微切割样品中提取 RNA 时，主要的影响因素有：样品的新旧程度、固定的过程和样品的储存条件。RNA 可能会降解成 <300 bp 的片段，而本试剂盒能有效吸附 >200 bp 的片段，所以如果样品中的 RNA 高度降解，可能会出现提不出 RNA 的情况。
1. 向样品中加入适当体积的 Buffer URL（使用前请先加入 β-巯基乙醇，终浓度为 1%），加入 Buffer URL 体积与微切割仪器的容积有关，但不能超过 140 µL。如有需要，可以将样品和 Buffer URL 转移至 1.5 mL 或 2 mL 的 RNase-Free 的离心管中（客户自备）。补充 Buffer URL 至体积为 150 µL。
注意：当处理的细胞量小于 5000 时，向溶液中加入 5 μL Trace RNA Enhancer。
2. 向溶液中加入 295 µL RNase-Free ddH₂O，然后加入 5 μL Proteinase K，用移液器混匀，55℃ 孵育 10 min。室温 12,000 rpm（13,680 × g）离心 3 min。
注意：此时的组织碎片可能会形成小球，有时在溶液上面会浮有一层薄膜。
3. 用移液器将上层溶液（450 µL 左右）转移至一个新的 RNase-Free 离心管中（客户自备）。
注意：使用移液器吸取上层溶液时，一定要避免接触到组织碎片小球，而且枪头要伸到薄膜以下进行吸取，避免将薄膜转移至离心管中。
4. 向吸出的上层溶液中加入 0.5 倍体积的无水乙醇（通常为 225 µL），用移液器混匀。将所有溶液及沉淀全部转移至吸附柱 RM 上（吸附柱放在收集管中），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
注意：在加入乙醇后，溶液中可能会出现沉淀，但是不会影响 RNA 的提取。
5. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
6. DNase I 工作液的配制：取 10 μL DNase I 储存液放入新的 RNase-Free 离心管中，加入 70 μL DNase I Buffer，轻柔混匀。
7. 向吸附柱中央加入 80 μL 的 DNase I 工作液，室温放置 15 min。
8. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
9. 向吸附柱中加入 500 μL Buffer RW2（使用前请先加入无水乙醇），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
10. 重复步骤 9。
11. 12,000 rpm（13,680 × g）离心 2 min。
12. 将吸附柱放入一个新的 1.5 mL RNase-Free 离心管中，打开吸附柱管盖，室温放置 1 min，以彻底晾干吸附柱中的无水乙醇。向吸附膜中央加入 10–15 μL RNase-Free ddH₂O，室温放置 1 min，12,000 rpm（13,680 × g）离心 30 sec，得到 RNA 溶液。
注意：洗脱体积不得少于 10 µL，否则会影响总的 RNA 得率。
二、从动物组织中提取总RNA
以下操作适用于从大多数动物组织中提取 RNA，如果要从纤维组织中提取 RNA，请参照三、从纤维组织中提取总RNA。从动物组织中提取 RNA，若要得到高纯度和高产量的 RNA，组织的起始量非常重要，裂解液最多能裂解 5 mg 的组织。有些组织像脾脏、部分脑组织、肺组织和胸腺组织在提取的过程中有可能会形成一些沉淀，但不会影响 RNA 的提取。
推荐使用的组织量不超过 5 mg，请不要超过吸附柱 RM 的载量，否则将会降低 RNA 的产量和纯度。
1. 确定组织的总量，不要超过 5 mg，立即进行第 2 步。
2. 在 Buffer URL（使用前请先加入 β-巯基乙醇，终浓度为 1%）中将组织块匀浆（2a）或者研碎（2b）。
注意：组织块的切碎和匀浆过程可采用下列 2a 和 2b 两种方法之一，当处理小于 10 µg 的组织时，需要向裂解液中加入 5 μL Trace RNA Enhancer。匀浆不彻底将导致 RNA 提取量低，还可能导致堵塞吸附柱的现象发生。
(2a) 使用匀浆器进行匀浆：
将称重过的组织块放在一个大小合适的容器中，加入 350 µL Buffer URL，立即进行匀浆至均一溶液（通常时间为 20–40 sec），继续步骤 3。
(2b) 使用研磨杵进行研磨：
在 1.5 mL RNase-Free 的离心管中（客户自备）加入 350 µL Buffer URL，将组织块迅速从 -80℃ 转入这个离心管中，用研磨杵充分研磨，彻底将组织破碎，注意避免组织冻融。将此溶液移至过滤柱 GS（客户自备）上（过滤柱放在收集管中），12,000 rpm（13,680 × g）离心 2 min，收集滤液，继续步骤 3。
注意：当使用研磨杵研磨时，请务必将组织彻底研碎，以保证裂解充分。
3. 将匀浆好的样品（2a）或者过滤液（2b）以 12,000 rpm（13,680 × g）离心 3 min，用移液器仔细将上清液转移到新的 1.5 mL RNase-Free 离心管中（客户自备）。
4. 向上清中加入 1 倍体积（350 µL）70% 乙醇，用移液器混匀，将所有溶液及沉淀全部转移至吸附柱 RM 上（吸附柱放在收集管中），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
注意：如果在以上过程中损失了一些裂解液，70% 乙醇的加量也要相应减少。在乙醇加入后，溶液中可能会出现沉淀，但不会影响 RNA 的提取。
5. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
6. DNase I 工作液的配制：取 10 μL DNase I 储存液放入新的 RNase-Free 离心管中，加入 70 μL DNase I Buffer，轻柔混匀。
7. 向吸附柱中央加入 80 μL 的 DNase I 工作液，室温放置 15 min。
8. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
9. 向吸附柱中加入 500 μL Buffer RW2（使用前请先加入无水乙醇），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
10. 重复步骤 9。
11. 12,000 rpm（13,680 × g）离心 2 min。
12. 将吸附柱放入一个新的 1.5 mL RNase-Free 离心管中，打开吸附柱管盖，室温放置 1 min，以彻底晾干吸附柱中的无水乙醇。向吸附膜中央加入 10–15 μL RNase-Free ddH₂O，室温放置 1 min，12,000 rpm（13,680 × g）离心 30 sec，得到 RNA 溶液。
注意：洗脱体积不得少于 10 µL，否则会影响总的 RNA 得率。
三、从纤维组织中提取总RNA
纤维组织，例如骨骼肌、心脏、皮肤中含有大量的收缩蛋白、连接组织和胶原质，只有去除这些蛋白质，才能使 RNA 的提取顺利进行。所以在从纤维组织中提取总 RNA 时，引入了蛋白酶 K 消化这一过程。
采用以下操作，可成功地从心脏、肌肉和皮肤组织中提取 RNA，其他富含蛋白质的组织也可采用此方法进行 RNA 的提取，值得注意的是，在进行蛋白酶 K 消化过程中的缓冲液不能对 RNase 进行长期有效的抑制，因而此方法不适用于含有丰富 RNase 的脾脏和肠组织等 RNA 的提取。
推荐使用的组织量不超过 5 mg，请不要超过吸附柱的载量，否则将会降低 RNA 的产量和纯度。
1. 确定组织的总量，不要超过 5 mg，立即进行第 2 步。
2. 在 Buffer URL（使用前请先加入 β-巯基乙醇，终浓度为 1%）中将组织块匀浆（2a）或者研碎（2b）。
注意：当处理小于 10 µg 的组织时，需要向裂解液中加入 5 μL Trace RNA Enhancer。匀浆不彻底会导致 RNA 提取量低，还可能导致吸附柱堵塞。采用匀浆器或过滤柱进行匀浆，会比采用其他方式匀浆获得更高的 RNA 得率。
(2a) 使用匀浆器进行匀浆：
将称重过的组织块放在一个大小合适的容器中，加入 150 µL Buffer URL，立即进行匀浆至均一溶液（通常时间为 20–40 sec），继续步骤 3。
(2b) 使用研磨杵进行研磨：
在 1.5 mL RNase-Free 的离心管中（客户自备）加入 150 µL Buffer URL，将组织块迅速从 -80℃ 转入这个离心管中，用研磨杵充分研磨，彻底将组织研碎，注意避免组织冻融，继续步骤 3。
注意：当使用研磨杵研磨时，请务必将组织彻底研碎，以保证裂解充分。
3. 向溶液中加入 295 µL RNase-Free ddH₂O，然后加入 5 µL Proteinase K，用移液器混匀。55℃ 孵育 10 min。室温 12,000 rpm（13,680 × g）离心 3 min。
注意：此时的组织碎片可能会形成小球，有时在溶液上面会浮有一层薄膜。
4. 用移液器将上层溶液（通常 450 µL 左右）转移至一个新的 1.5 mL RNase-Free 的离心管中（客户自备）。
注意：使用移液器吸取上层溶液时，一定要避免接触到组织碎片小球，而且枪头要伸到薄膜以下进行吸取，避免将薄膜转移至离心管中。
5. 向吸出的上层溶液加入 0.5 倍体积的无水乙醇（通常为 225 µL），用移液器混匀。将所有溶液及沉淀物质全部转移至吸附柱 RM 上（吸附柱放在收集管中），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
注意：在乙醇加入后，溶液中可能会出现沉淀，但是不会影响 RNA 的提取。
6. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
7. DNase I 工作液的配制：取 10 μL DNase I 储存液放入新的 RNase-Free 离心管中，加入 70 μL DNase I Buffer，轻柔混匀。
8. 向吸附柱中央加入 80 μL 的 DNase I 工作液，室温放置 15 min。
9. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
10. 向吸附柱中加入 500 μL Buffer RW2（使用前请先加入无水乙醇），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
11. 重复步骤 10。
12. 12,000 rpm（13,680 × g）离心 2 min。
13. 将吸附柱放入一个新的 1.5 mL RNase-Free 离心管中，打开吸附柱管盖，室温放置 1 min，以彻底晾干吸附柱中的无水乙醇。向吸附膜中央加入 10–15 μL RNase-Free ddH₂O，室温放置 1 min，12,000 rpm（13,680 × g）离心 30 sec，得到 RNA 溶液。
注意：洗脱体积不得少于 10 µL，否则会影响总的 RNA 得率。
四、从动物细胞中提取总RNA
推荐使用的细胞量不超过 5×10⁵，请不要超过吸附柱的载量，否则将会降低 RNA 的产量和纯度。
1. 细胞的处理和裂解
1.1 细胞沉淀
轻弹管底使细胞分开，加入 350 µL Buffer URL（使用前请先加入 β-巯基乙醇，终浓度为 1%），涡旋振荡或用移液器混匀，继续步骤 2。
1.2 悬浮培养细胞
确定细胞的数量（见表 2），在 RNase-Free 的离心管（客户自备）中 300×g 离心 5 min 沉淀细胞，去除所有上清，加入 350 µl Buffer URL（使用前请先加入 β-巯基乙醇，终浓度为 1%），继续步骤 2。
1.3 单层贴壁细胞
对于该种细胞的收集可直接在细胞培养器皿中进行（直径最大为 10 cm），或者可以先用胰蛋白酶消化，再收集细胞沉淀，进行裂解。
- 直接在细胞培养器皿中进行裂解：
确定细胞数量，将细胞培养基完全吸出，加入 350 µL Buffer URL（使用前请先加入 β-巯基乙醇，终浓度为 1%）裂解细胞，收集细胞裂解液并将其转移至 1.5 mL RNase-Free 离心管（客户自备）中，涡旋振荡或用移液器混匀，确保看不见任何成团的细胞之后，继续步骤 2。 - 胰蛋白酶消化（摇瓶中培养的单层贴壁细胞，通常采用胰蛋白酶消化的方法）：
确定细胞数量，吸出培养基，用 PBS 缓冲液洗细胞，吸除 PBS 缓冲液，加入含有 0.10%—0.25% 胰蛋白酶的 PBS 缓冲液处理细胞，在细胞脱离容器壁后，加入含有血清的溶液使胰蛋白酶失活，将细胞转移到 RNase-Free 的离心管中，300×g 离心 5 min 沉淀细胞，仔细去除所有上清。加入 350 µL Buffer URL（使用前请先加入 β-巯基乙醇，终浓度为 1%）裂解细胞。继续步骤 2。
注意：
① 如果细胞培养基没有去除干净，将会稀释裂解液，影响裂解效果及 RNA 与硅胶膜的结合，这将导致 RNA 的提取量降低。
② 当处理的细胞量 ≤1×10⁵ 时，Buffer URL 的加量可以减少至 75 µL，这时可采用小一些的离心管，涡旋振荡 1 min 使裂解液匀质化，进行步骤 2。
③ 如果处理细胞量 <5000 时，需要向裂解液中加入 5 μL Trace RNA Enhancer。
④ 如果细胞重悬不完全，将会导致裂解不充分，降低 RNA 的产率。
2. 样品的匀质化（请见步骤 2a，2b）
注意：匀质不彻底将导致 RNA 提取量低，还可能导致吸附柱 CR1 堵塞，采用匀浆器或过滤柱进行匀质，会比采用其他方式匀浆获得更高的 RNA 得率。
(2a) 将加入裂解液的细胞转移至过滤柱 GS（客户自备）上（过滤柱放在收集管中），12,000 rpm（13,680 × g）离心 2 min，收集滤液，继续步骤 3。
(2b) 使用匀浆器匀浆 30 sec。
3. 加入 1 倍体积（通常为 350 µL）70% 乙醇，用移液器混匀。将所有溶液及沉淀全部转移至吸附柱 RM 上（吸附柱放在收集管中），12,000 rpm（13,680 × g）离心 30–60 sec，弃废液，将吸附柱放回收集管中。
注意：如果在匀质的过程中损失了一些裂解液，70% 乙醇的加量也要相应减少。如果在第 2 步中只加入了 75 µL 的 Buffer URL，则在本步骤中也只需加入 75 µL 的 70% 乙醇，加入乙醇后，溶液中可能会出现沉淀，但是不会影响 RNA 的提取。
4. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
5. DNase I 工作液的配制：取 10 μL DNase I 储存液放入新的 RNase-Free 离心管中，加入 70 μL DNase I Buffer，轻柔混匀。
6. 向吸附柱中央加入 80 μL 的 DNase I 工作液，室温放置 15 min。
7. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
8. 向吸附柱中加入 500 μL Buffer RW2（使用前请先加入无水乙醇），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
9. 重复步骤 8。
10. 12,000 rpm（13,680 × g）离心 2 min。
11. 将吸附柱放入一个新的 1.5 mL RNase-Free 离心管中，打开吸附柱管盖，室温放置 1 min，以彻底晾干吸附柱中的无水乙醇。向吸附膜中央加入 10–15 μL RNase-Free ddH₂O，室温放置 1 min，12,000 rpm（13,680 × g）离心 30 sec，得到 RNA 溶液。
注意：洗脱体积不得少于 10 µL，否则会影响总的 RNA 得率。
This product uses new technology to extract RNA from micro amounts of tissues and cells (as few as 10 cells). Special components added to the lysis buffer greatly increase the selective binding ability of RNA to the silica membrane. During the RNA extraction process, DNase I is added to completely remove trace DNA impurities, while DNase I and other residual substances are removed in subsequent washing steps, effectively ensuring RNA yield and purity.
Compared with ordinary RNA extraction methods, the Micro Sample RNA Extraction Kit selectively removes all RNA <200 bp (5.8S rRNA, 5S rRNA, tRNAs, etc.), while RNA >200 bp is enriched, separated, and purified. In this manual, different sample types have different procedures. The differences mainly lie in sample lysis and binding conditions to the silica membrane. Once RNA binds to the membrane, the subsequent steps are essentially similar.
Precautions
1. Reagents and consumables to be prepared by the user:
β‑mercaptoethanol, absolute ethanol, 1.5 mL RNase‑Free centrifuge tubes.
2. Determining sample amount
To obtain high yield and purity using this kit, appropriate sample amount is important because the binding capacity of the spin column and the volume of lysis buffer are limited. Only when the sample is fully lysed and RNA is adequately bound to the silica membrane can the RNA yield be ensured.
Table 1. Technical specifications of the Micro Sample RNA Extraction Kit
|
Note: If the maximum binding capacity of the kit is exceeded, RNA yield may decrease. If the sample is not completely lysed, RNA yield will also decrease.
Table 2. Number of HeLa cells under different culture conditions
|
3. Sample storage and handling
RNA in untreated tissue is unprotected and easily degraded. Fresh tissue should be quickly frozen in liquid nitrogen and stored immediately at -70°C. Frozen tissue should not be repeatedly freeze‑thawed to avoid RNA degradation. Samples can also be homogenized, added to Buffer URL, and stored at -70°C; frozen samples can be stored stably for several months.
Pre‑operation Precautions
To prevent RNA degradation, avoid leaving untreated samples at room temperature for extended periods. Before tissue fixation, RNA is unprotected, so freeze the tissue in liquid nitrogen.
Tissue stored in Buffer URL can be stored at -70°C for several months. When processing frozen lysates, thaw samples completely at room temperature or in a 37°C water bath. Ensure that the salt in the lysate is completely dissolved, then proceed immediately (prolonged 37°C treatment may cause chemical degradation of RNA).
Before use, prepare 70% ethanol using RNase‑Free ddH₂O.
Preparation of DNase I stock solution: Use a 1 mL syringe to inject 550 μL RNase‑Free ddH₂O into the vial containing DNase I powder. Mix gently until dissolved. Aliquot and store at -30 to -15°C (can be stored for 9 months). Thaw aliquots as needed. The prepared DNase I working solution can be stored at 2‑8°C for up to 42 days. Avoid repeated freeze‑thaw cycles.
Protocol
Preparation before use: Add 48 mL of absolute ethanol to 12 mL of Buffer RW2.
Total RNA Extraction from Formalin‑Fixed Microdissected Samples
When extracting RNA from formalin‑fixed microdissected samples, the main influencing factors are sample age, fixation process, and storage conditions. RNA may be degraded into fragments <300 bp. This kit efficiently binds fragments >200 bp, so if the RNA in the sample is highly degraded, extraction may fail.
1. Add an appropriate volume of Buffer URL (add β‑mercaptoethanol to a final concentration of 1% before use) to the sample. The volume of Buffer URL added depends on the capacity of the microdissection instrument, but should not exceed 140 µL. If needed, transfer the sample and Buffer URL to a 1.5 mL or 2 mL RNase‑Free centrifuge tube (user‑supplied). Add Buffer URL to bring the total volume to 150 µL.
Note: When processing less than 5000 cells, add 5 μL Trace RNA Enhancer to the solution.
2. Add 295 µL RNase‑Free ddH₂O to the solution, then add 5 μL Proteinase K. Mix with a pipette and incubate at 55°C for 10 minutes. Centrifuge at room temperature at 12,000 rpm (13,680 × g) for 3 minutes.
Note: Tissue debris may form a pellet, and sometimes a thin film may float on top of the solution.
3. Transfer the upper solution (approximately 450 µL) to a new RNase‑Free centrifuge tube (user‑supplied) using a pipette.
Note: When aspirating the upper solution, be careful to avoid touching the tissue debris pellet. Insert the pipette tip below the film to avoid transferring the film to the new tube.
4. Add 0.5 volumes of absolute ethanol (usually 225 µL) to the transferred upper solution and mix with a pipette. Transfer all solution and precipitate to a Spin Column RM (placed in a collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
Note: After adding ethanol, a precipitate may appear, but this does not affect RNA extraction.
5. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
6. Preparation of DNase I working solution: Transfer 10 μL of DNase I stock solution to a new RNase‑Free centrifuge tube, add 70 μL DNase I Buffer, and mix gently.
7. Add 80 μL of the DNase I working solution to the center of the column. Let stand at room temperature for 15 minutes.
8. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
9. Add 500 μL Buffer RW2 (ensure absolute ethanol has been added before use) to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
10. Repeat step 9.
11. Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes.
12. Transfer the column to a new 1.5 mL RNase‑Free centrifuge tube. Open the lid and let stand at room temperature for 1 minute to completely air‑dry any residual ethanol. Add 10–15 μL RNase‑Free ddH₂O to the center of the membrane. Let stand at room temperature for 1 minute, then centrifuge at 12,000 rpm (13,680 × g) for 30 seconds to collect the RNA solution.
Note: The elution volume should not be less than 10 µL, otherwise total RNA yield may be affected.
Total RNA Extraction from Animal Tissue
The following procedure is suitable for RNA extraction from most animal tissues. For fibrous tissues, refer to Section III (Total RNA Extraction from Fibrous Tissue). For high‑purity and high‑yield RNA from animal tissue, the starting amount is critical. The lysis buffer can lyse up to 5 mg of tissue. Some tissues (spleen, certain brain regions, lung, thymus) may form precipitates during extraction, but this does not affect RNA extraction.
Recommended tissue amount: ≤5 mg. Do not exceed the binding capacity of the Spin Column RM, otherwise RNA yield and purity may decrease.
1. Determine the total tissue amount (not exceeding 5 mg) and proceed immediately to step 2.
2. Homogenize (2a) or grind (2b) the tissue piece in Buffer URL (add β‑mercaptoethanol to a final concentration of 1% before use).
Note: When processing less than 10 µg of tissue, add 5 μL Trace RNA Enhancer to the lysis buffer. Incomplete homogenization may lead to low RNA yield and column clogging.
(2a) Homogenization using a homogenizer:
Place the weighed tissue piece into a suitable container, add 350 µL Buffer URL, and immediately homogenize until a uniform solution is obtained (typically 20–40 seconds). Proceed to step 3.
(2b) Grinding using a pestle:
Add 350 µL Buffer URL to a 1.5 mL RNase‑Free centrifuge tube (user‑supplied). Quickly transfer the tissue piece from -80°C into the tube. Grind thoroughly with a pestle to completely disrupt the tissue, avoiding freeze‑thaw. Transfer the solution to a Filter Column GS (user‑supplied, placed in a collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes. Collect the flow‑through. Proceed to step 3.
Note: When using a pestle, ensure the tissue is completely ground to achieve full lysis.
3. Centrifuge the homogenized sample (2a) or filtrate (2b) at 12,000 rpm (13,680 × g) for 3 minutes. Carefully transfer the supernatant to a new 1.5 mL RNase‑Free centrifuge tube (user‑supplied) using a pipette.
4. Add 1 volume (350 µL) of 70% ethanol to the supernatant and mix with a pipette. Transfer all solution and precipitate to a Spin Column RM (placed in a collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
Note: If some lysis buffer was lost during previous steps, reduce the amount of 70% ethanol accordingly. After adding ethanol, a precipitate may appear, but this does not affect RNA extraction.
5. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
6. Preparation of DNase I working solution: Transfer 10 μL of DNase I stock solution to a new RNase‑Free centrifuge tube, add 70 μL DNase I Buffer, and mix gently.
7. Add 80 μL of the DNase I working solution to the center of the column. Let stand at room temperature for 15 minutes.
8. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
9. Add 500 μL Buffer RW2 (ensure absolute ethanol has been added before use) to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
10. Repeat step 9.
11. Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes.
12. Transfer the column to a new 1.5 mL RNase‑Free centrifuge tube. Open the lid and let stand at room temperature for 1 minute to completely air‑dry any residual ethanol. Add 10–15 μL RNase‑Free ddH₂O to the center of the membrane. Let stand at room temperature for 1 minute, then centrifuge at 12,000 rpm (13,680 × g) for 30 seconds to collect the RNA solution.
Note: The elution volume should not be less than 10 µL, otherwise total RNA yield may be affected.
Total RNA Extraction from Fibrous Tissue
Fibrous tissues (e.g., skeletal muscle, heart, skin) contain large amounts of contractile proteins, connective tissue, and collagen. Removal of these proteins is necessary for successful RNA extraction. Therefore, for total RNA extraction from fibrous tissue, a Proteinase K digestion step is introduced.
The following procedure can successfully extract RNA from heart, muscle, and skin tissues. Other protein‑rich tissues can also use this method. However, the buffer used during Proteinase K digestion does not provide long‑term effective RNase inhibition, so this method is not suitable for RNA extraction from RNase‑rich tissues such as spleen and intestine.
Recommended tissue amount: ≤5 mg. Do not exceed the binding capacity of the spin column, otherwise RNA yield and purity may decrease.
1. Determine the total tissue amount (not exceeding 5 mg) and proceed immediately to step 2.
2. Homogenize (2a) or grind (2b) the tissue piece in Buffer URL (add β‑mercaptoethanol to a final concentration of 1% before use).
Note: When processing less than 10 µg of tissue, add 5 μL Trace RNA Enhancer to the lysis buffer. Incomplete homogenization may lead to low RNA yield and column clogging. Using a homogenizer or filter column yields higher RNA recovery than other methods.
(2a) Homogenization using a homogenizer:
Place the weighed tissue piece into a suitable container, add 150 µL Buffer URL, and immediately homogenize until a uniform solution is obtained (typically 20–40 seconds). Proceed to step 3.
(2b) Grinding using a pestle:
Add 150 µL Buffer URL to a 1.5 mL RNase‑Free centrifuge tube (user‑supplied). Quickly transfer the tissue piece from -80°C into the tube. Grind thoroughly with a pestle to completely disrupt the tissue, avoiding freeze‑thaw. Proceed to step 3.
Note: When using a pestle, ensure the tissue is completely ground to achieve full lysis.
3. Add 295 µL RNase‑Free ddH₂O to the solution, then add 5 μL Proteinase K. Mix with a pipette. Incubate at 55°C for 10 minutes. Centrifuge at room temperature at 12,000 rpm (13,680 × g) for 3 minutes.
Note: Tissue debris may form a pellet, and sometimes a thin film may float on top of the solution.
4. Transfer the upper solution (usually about 450 µL) to a new 1.5 mL RNase‑Free centrifuge tube (user‑supplied) using a pipette.
Note: When aspirating the upper solution, be careful to avoid touching the tissue debris pellet. Insert the pipette tip below the film to avoid transferring the film to the new tube.
5. Add 0.5 volumes of absolute ethanol (usually 225 µL) to the transferred upper solution and mix with a pipette. Transfer all solution and precipitate to a Spin Column RM (placed in a collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
Note: After adding ethanol, a precipitate may appear, but this does not affect RNA extraction.
6. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
7. Preparation of DNase I working solution: Transfer 10 μL of DNase I stock solution to a new RNase‑Free centrifuge tube, add 70 μL DNase I Buffer, and mix gently.
8. Add 80 μL of the DNase I working solution to the center of the column. Let stand at room temperature for 15 minutes.
9. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
10. Add 500 μL Buffer RW2 (ensure absolute ethanol has been added before use) to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
11. Repeat step 10.
12. Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes.
13. Transfer the column to a new 1.5 mL RNase‑Free centrifuge tube. Open the lid and let stand at room temperature for 1 minute to completely air‑dry any residual ethanol. Add 10–15 μL RNase‑Free ddH₂O to the center of the membrane. Let stand at room temperature for 1 minute, then centrifuge at 12,000 rpm (13,680 × g) for 30 seconds to collect the RNA solution.
Note: The elution volume should not be less than 10 µL, otherwise total RNA yield may be affected.
Total RNA Extraction from Animal Cells
Recommended cell amount: ≤5×10⁵. Do not exceed the binding capacity of the spin column, otherwise RNA yield and purity may decrease.
1. Cell processing and lysis
1.1 Cell pellet
Gently tap the bottom of the tube to disperse the cells. Add 350 µL Buffer URL (add β‑mercaptoethanol to a final concentration of 1% before use). Vortex or mix with a pipette. Proceed to step 2.
1.2 Suspension culture cells
Determine the cell number (see Table 2). Pellet the cells by centrifugation at 300×g for 5 minutes in an RNase‑Free centrifuge tube (user‑supplied). Remove all supernatant. Add 350 µL Buffer URL (add β‑mercaptoethanol to a final concentration of 1% before use). Proceed to step 2.
1.3 Monolayer adherent cells
These cells can be collected directly in the culture vessel (maximum diameter 10 cm), or they can be trypsinized and then pelleted for lysis.
Direct lysis in the culture vessel:
Determine the cell number. Completely remove the culture medium. Add 350 µL Buffer URL (add β‑mercaptoethanol to a final concentration of 1% before use) to lyse the cells. Collect the cell lysate and transfer to a 1.5 mL RNase‑Free centrifuge tube (user‑supplied). Vortex or mix with a pipette until no cell clumps are visible. Proceed to step 2.
Trypsinization (for monolayer adherent cells grown in flasks):
Determine the cell number. Aspirate the medium, wash cells with PBS, aspirate PBS, and treat cells with PBS containing 0.10%–0.25% trypsin. When cells detach from the vessel wall, add serum‑containing medium to inactivate trypsin. Transfer cells to an RNase‑Free centrifuge tube. Pellet cells by centrifugation at 300×g for 5 minutes. Carefully remove all supernatant. Add 350 µL Buffer URL (add β‑mercaptoethanol to a final concentration of 1% before use) to lyse the cells. Proceed to step 2.
Notes:
① If culture medium is not completely removed, it will dilute the lysis buffer, affecting lysis efficiency and RNA binding to the silica membrane, leading to reduced RNA yield.
② When processing ≤1×10⁵ cells, the volume of Buffer URL can be reduced to 75 µL. Use a smaller centrifuge tube and vortex for 1 minute to homogenize the lysate. Then proceed to step 2.
③ If processing <5000 cells, add 5 μL Trace RNA Enhancer to the lysis buffer.
④ Incomplete resuspension of cells will lead to insufficient lysis and reduced RNA yield.
2. Sample homogenization (see steps 2a, 2b)
Note: Incomplete homogenization may lead to low RNA yield and clogging of the Spin Column CR1. Using a homogenizer or filter column yields higher RNA recovery than other methods.
(2a) Transfer the cell lysate to a Filter Column GS (user‑supplied, placed in a collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes. Collect the flow‑through. Proceed to step 3.
(2b) Homogenize using a homogenizer for 30 seconds.
3. Add 1 volume (usually 350 µL) of 70% ethanol and mix with a pipette. Transfer all solution and precipitate to a Spin Column RM (placed in a collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 30–60 seconds. Discard the flow‑through and return the column to the collection tube.
Note: If some lysis buffer was lost during homogenization, reduce the amount of 70% ethanol accordingly. If only 75 µL of Buffer URL was added in step 2, then add only 75 µL of 70% ethanol here. After adding ethanol, a precipitate may appear, but this does not affect RNA extraction.
4. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
5. Preparation of DNase I working solution: Transfer 10 μL of DNase I stock solution to a new RNase‑Free centrifuge tube, add 70 μL DNase I Buffer, and mix gently.
6. Add 80 μL of the DNase I working solution to the center of the column. Let stand at room temperature for 15 minutes.
7. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
8. Add 500 μL Buffer RW2 (ensure absolute ethanol has been added before use) to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
9. Repeat step 8.
10. Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes.
11. Transfer the column to a new 1.5 mL RNase‑Free centrifuge tube. Open the lid and let stand at room temperature for 1 minute to completely air‑dry any residual ethanol. Add 10–15 μL RNase‑Free ddH₂O to the center of the membrane. Let stand at room temperature for 1 minute, then centrifuge at 12,000 rpm (13,680 × g) for 30 seconds to collect the RNA solution.
Note: The elution volume should not be less than 10 µL, otherwise total RNA yield may be affected.
规格
别名
微量RNA快速提取试剂盒
英文别名
Total RNA Micro Kit
规格或纯度
BioReagent,分子生物学级
英文名称
Micro Sample Total RNA Extraction Kit (DNase I)
应用
RNA Extraction
储存与运输
储存条件
2-8°C储存,室温,-20°C储存
运输条件
超低温运输
稳定性与储存
各组分在相应的储存条件下保质期15个月。
组分与储存
| T1521420 | 组件名称 | 50T | 储存温度 |
| T1521420A | Buffer URL | 30 mL | RT. |
| T1521420B | Buffer RW | 38 mL | RT. |
| T1521420C | Buffer RW2 | 12 mL | RT. |
| T1521420D | RNase-Free ddH₂O | 15 mL | RT. |
| T1521420E | Proteinase K | 0.7 mL | RT. |
| T1521420F | 吸附柱RM (含2mL收集管) | 1 EA×50 | RT. |
| T1521420G | RNase-Free离心管 (1.5mL) | 1 EA×50 | RT. |
| T1521420H | 1mL注射器 | 1 EA | RT. |
| T1521420I | DNase I | 1 EA | 2-8℃. |
| T1521420J | DNase I Buffer | 4 mL | 2-8℃. |
| T1521420K | RNase-Free ddH₂O | 1 mL | 2-8℃. |
| T1521420L | Trace RNA Enhancer | 280 μL | -20℃. |
T1521420A, T1521420B, T1521420C, T1521420D, T1521420E, T1521420F, T1521420G, T1521420H：室温保存，15个月。
T1521420I, T1521420J, T1521420K：2-8℃保存，15个月。
T1521420L：4℃可存放1个月，长期置于-20℃保存。
加入β-巯基乙醇的Buffer URL可在4℃放置1个月。
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0636699 |
分析证书 | T1521420 |
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溶液计算器
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