基本描述

规格或纯度

BioReagent, 分子生物学级, 无RNA酶, for DNA and RNA applications

稳定性与储存

Store at room temperature long term (12 months).

英文名称

RNApure Blood Kit

储存温度

室温

运输条件

常规运输

产品介绍

本试剂盒适用于从新鲜全血（用柠檬酸盐、EDTA或肝素等抗凝剂处理过的血液样品）中提取总RNA，可以处理多至1.5 ml的全血，洗脱得到分子量大于200 bp的高纯度 RNA，多个样品可以在1小时内同时完成。本产品无需CsCl纯化的超离心步骤和LiCl或乙醇沉淀，不含有苯酚或氯仿等有毒溶剂，经过纯化的RNA有效去除血红素、肝素等酶抑制剂和污染物，可直接用于各种分子生物学常规实验，如RT-PCR、Northern Blot、 Dot Blot、体外翻译等实验。

R666034	Component	50 T	Storage
R666034A	Buffer RBL (10×)	60 mL	RT
R666034B	Buffer RL	35 mL	RT
R666034C	Buffer RW1	40 mL	RT
R666034D	Buffer RW2 (concentrate)	11 mL	RT
R666034E	RNase-Free Water	10 mL	RT
R666034F	Spin Columns FL with Collection Tubes	50 sets	RT
R666034G	Spin Columns RM with Collection Tubes	50 sets	RT
R666034H	RNase-Free Centrifuge Tubes (1.5 mL)	50 EA	RT

自备试剂：β-巯基乙醇、70%乙醇（用无RNase的水配制）、无水乙醇。

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0.5M 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．样品应避免反复冻融，否则影响RNA提取得率和质量。样品在Buffer RL中，可于-70℃ 保存一个月。

3．使用前请检查Buffer RL是否出现结晶或者沉淀，可置于56℃水浴重新溶解。Buffer RL 在使用前请加入β-巯基乙醇，至终浓度为1%。如1 ml Buffer RL加10 μl β-巯基乙醇。加入β-巯基乙醇的Buffer RL室温可保存1个月。

4．第一次使用前应按照试剂瓶标签的说明先在Buffer RW2中加入无水乙醇。

5．本试剂盒不能用于加入抗凝剂的冷冻血液样本RNA的提取。

6．10×Buffer RBL需在使用前将溶液用无RNase的水进行10倍稀释，稀释后置于2-8℃ 保存。

7．若下游实验对DNA非常敏感，建议用不含RNase的DNase I对 RNA进行处理。

8．所有离心步骤如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

1．向0.5-1.5 ml 新鲜的抗凝全血样本中，加入5倍体积的1×Buffer RBL（请于使用前用无RNase的水将10×Buffer RBL稀释10倍），轻轻涡旋或颠倒混匀。冰上孵育10-15 分钟，孵育过程中混匀两次。

注意：孵育过程中浑浊的悬液会变成透明，证明红细胞被裂解，必要时可以把孵育时间延长至20分钟。

2．4℃ 2,100 rpm（~400×g）离心10分钟，小心吸弃上清。

3．向以上沉淀中加入2倍血液样本体积的1×Buffer RBL（请于使用前用无RNase的水将10×Buffer RBL稀释10倍），轻轻涡旋，充分重悬沉淀。

4．4℃ 2,100 rpm离心10分钟，小心并彻底吸弃上清。

注意：此步一定要完全去除上清否则影响裂解导致RNA产量下降。

5．向沉淀中加入Buffer RL（使用前检查是否已经加入β-巯基乙醇），0.5-1.5 ml血液样本加入600 μl Buffer RL，或小于0.5 ml 血液样本加入350 μl Buffer RL，混匀。

6．将所得液体转移到已装入收集管的过滤柱（Spin Columns FL）中，12,000 rpm （~13,400×g）离心2分钟，收集滤液，弃掉过滤柱。

7．向所得滤液中加入1倍体积（600 μl或350 μl）的70%乙醇（无RNase水配制），混匀。

注意：加入乙醇后可能会产生沉淀，不会影响后续实验。

8．将上步所得溶液全部加入到已装入收集管的吸附柱（Spin Columns RM）中，若一次不能加完溶液，可分多次转入。12,000 rpm离心15秒，倒掉收集管中的废液，将吸附柱重新放回收集管中。

9．向吸附柱中加入700 μl Buffer RW1，12,000 rpm离心15秒，倒掉收集管中的废液，将吸附柱重新放回收集管中。

可选步骤：如果要进行对微量DNA非常敏感的RNA实验，则用以下步骤替代步骤9。

1）向吸附柱中加入350 μl Buffer RW1, 12,000 rpm离心15秒，倒掉收集管中的废液，将吸附柱重新放回收集管中。

2）配制DNase I 混合液：取70 μl Reaction Buffer 和10 μl DNase I 储存液，轻柔混匀，配制成终体积为80 μl的反应液。

注意: 以上体系为按照我公司产品DNase I （D665537）反应体系进行配置，应用其他公司产品请参考相应说明书。

3）向吸附柱中直接加入80 µl 配制好的DNase I 反应液，20-30℃孵育15分钟。

4）向吸附柱中加入350 μl Buffer RW1, 12,000 rpm离心15秒，倒掉收集管中的废液，将吸附柱重新放回收集管中。

10．向吸附柱中加入500 μl Buffer RW2（使用前检查是否已加入无水乙醇），12,000 rpm 离心15秒，倒掉收集管中的废液，将吸附柱重新放回收集管中。

11．重复步骤10。

12．12,000 rpm离心2分钟，倒掉收集管中的废液。将吸附柱置于室温数分钟，以彻底晾干。

注意：这一步的目的是将吸附柱中残余的乙醇去除，乙醇的残留会影响后续的酶促反应（酶切、 PCR 等）。

13．将吸附柱置于一个新的无RNase离心管中，向吸附柱的中间部位加入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， -70℃保存RNA，防止降解。

注意：

1）RNase-Free Wate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中，重复步骤13。

This kit is suitable for extracting total RNA from fresh whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin). It can process up to 1.5 ml of whole blood and elute to obtain high-purity RNA with a molecular weight greater than 200 bp. Multiple samples can be completed simultaneously within 1 hour. This product does not require the ultra centrifugation step of CsCl purification and LiCl or ethanol precipitation. It does not contain toxic solvents such as phenol or chloroform. The purified RNA effectively removes enzyme inhibitors and pollutants such as heme and heparin. It can be directly used in various molecular biology routine experiments, such as RT-PCR, Northern Blot, Dot Blot, in vitro translation, and so on.

Self prepared reagents: β- Mercaptoethanol, 70% ethanol (prepared with water without RNase), anhydrous ethanol.

R666034	Component	50 T	Storage
R666034A	Buffer RBL (10×)	60 mL	RT
R666034B	Buffer RL	35 mL	RT
R666034C	Buffer RW1	40 mL	RT
R666034D	Buffer RW2 (concentrate)	11 mL	RT
R666034E	RNase-Free Water	10 mL	RT
R666034F	Spin Columns FL with Collection Tubes	50 sets	RT
R666034G	Spin Columns RM with Collection Tubes	50 sets	RT
R666034H	RNase-Free Centrifuge Tubes (1.5 mL)	50 EA	RT

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 0.5M 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 sample should avoid repeated freezing and thawing, otherwise it will affect the yield and quality of RNA extraction. The sample can be stored in Buffer RL at -70 ℃ for one month.
3. Before use, please check if there is any crystallization or precipitation in the Buffer RL. It can be dissolved again in a 56 ℃ water bath. Please add Buffer RL before use β- Mercaptoethanol, with a final concentration of 1%. Add 10 to 1 ml Buffer RL μ L β- Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month.
4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.
5. This reagent kit cannot be used for RNA extraction from frozen blood samples with anticoagulants added.
6.10 × Buffer RBL needs to be diluted 10 times with water without RNase before use, and then stored at 2-8 ℃ after dilution.
7. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I that does not contain RNase.
8. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.

Operation steps
1. Add 5 times the volume of 1 x Buffer RBL to fresh anticoagulant whole blood samples of 0.5-1.5 ml (please dilute 10 x Buffer RBL with RNase free water before use), gently vortex or invert and mix well. Incubate on ice for 10-15 minutes, mix twice during the incubation process.
Attention: During the incubation process, the cloudy suspension will become transparent, indicating that red blood cells have been lysed. If necessary, the incubation time can be extended to 20 minutes.
2. Centrifuge at 4 ℃, 2100 rpm (~400 × g) for 10 minutes, and carefully discard the supernatant.
3. Add 2 times the volume of the blood sample to the above precipitate with 1 x Buffer RBL (please dilute 10 x Buffer RBL with RNase free water before use), gently vortex, and resuspend the precipitate thoroughly.
4. Centrifuge at 4 ℃ and 2100 rpm for 10 minutes, carefully and thoroughly remove the supernatant.
Note: This step must completely remove the supernatant, otherwise it will affect the lysis and lead to a decrease in RNA production.
5. Add Buffer RL to the precipitate (check if it has been added before use β- Mercaptoethanol, 0.5-1.5 ml of blood sample added to 600 μ L Buffer RL, or less than 0.5 ml of blood sample added to 350 μ L Buffer RL, mix well.
6. Transfer the obtained liquid to the spin columns FL that have been loaded into the collection tube, centrifuge at 12000 rpm (~13400 × g) for 2 minutes, collect the filtrate, and discard the filter column.
7. Add 1 volume (600) to the obtained filtrate μ L or 350 μ l) Mix 70% ethanol (prepared without RNase water) well.
Attention: Adding ethanol may cause precipitation and will not affect subsequent experiments.
8. Add all the solution obtained in the previous step to the spin columns RM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred in multiple batches. Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
9. Add 700 to the adsorption column μ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
Optional steps: If conducting RNA experiments that are highly sensitive to trace amounts of DNA, replace step 9 with the following steps.

1) Add 350 to the adsorption column μ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
2) Preparation of DNase I mixture: Take 70 μ Reaction Buffer and 10 μ L DNase I storage solution, gently mix and prepare to a final volume of 80 μ The reaction solution of L.
Attention: The above system is configured according to our company's DNase I (D665537) reaction system. Please refer to the corresponding manual for other company products.
1) Add 350 to the adsorption column μ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
2) Preparation of DNase I mixture: Take 70 μ Reaction Buffer and 10 μ L DNase I storage solution, gently mix and prepare to a final volume of 80 μ The reaction solution of L.
Attention: The above system is configured according to our company's DNase I (D665537) reaction system. Please refer to the corresponding manual for other company products.
3) Add 80 µ l of the prepared DNase I reaction solution directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.
4) Add 350 to the adsorption column μ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
10. Add 500 to the adsorption column μ Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
11. Repeat step 10.
12. Centrifuge at 12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column 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, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
13. Place the adsorption column 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 RNA 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 and repeat step 13.