本试剂盒适合于从各种组织、细胞中分离纯化高质量的总RNA。试剂盒基于超顺磁性的磁性粒子纯化技术，无需进行耗时的醇类沉淀。得到的RNA可直接用于RT-PCR、Real Time RT-PCR、芯片分析、Northern Blot、Dot Blot、Poly A筛选、体外翻译、RNase保护分析和分子克隆等多种下游实验。其处理后的产物用于科研检测使用。
储存条件
M1521688A, M1521688C, M1521688E, M1521688F, M1521688G, M1521688J：室温保存，12个月。
M1521688B, M1521688D, M1521688I：2-8℃保存，12个月。
M1521688H：-20℃保存，12个月。
适配仪器
手工提取
注意事项
1. 使用前请仔细阅读本说明书。
2. 本试剂盒中的多种缓冲液含有刺激性的胍盐，务必戴上手套，并按照安全标准预防措施来处理。不要让缓冲液接触到皮肤、眼睛以及黏膜，如果确实发生，请立即用大量清水清洗并就医。
3. 如因气温较低，溶液出现沉淀，需要30℃水浴至沉淀完全溶解后方可使用。
4. 如因气温较低，用力振荡后磁珠无法重悬，请勿使用。
5. 本试剂盒中的多种缓冲液含胍盐，请勿用氧化性消毒剂如次氯酸钠进行处理，否则会释放有毒气体，须按医疗废物进行处理。
6. 本试剂盒需配套使用，不同批号试剂盒中各组分不可以互换或混用。
使用方法
1. 样本要求
- 适用标本类型：动物组织、4~6× 10⁵细胞等样本。
- 标本保存和运输：标本可立即用于测试，也可以保存于-70℃或更低温度待测，保存期为6个月，应避免反复冻融。标本运输采用冷链运输。
- 冻融要求：速冻速融，避免反复冻融。
2. 自备试验器材
- 恒温水浴锅（当溶液出现沉淀时使用）、干式恒温仪
- 磁力架（1.5/2.0 mL）、1.5/2.0 mL 无菌无核酸酶离心管、各种规格移液器及Tips（10、20、200、1000 μL）
3. 试剂准备
4. 操作步骤
磁分离操作建议（步骤4.3开始）：离心管置于磁力架后，轻轻地左右旋转，待磁珠聚集于贴近磁力架的管壁后，轻轻地颠倒磁力架数次，使管盖上的磁珠也聚集到管壁，静置数分钟至溶液澄清即可（静置时长视磁力架磁性而定）。
4.1 样本前处理
(1) 组织样本处理
取新鲜或-80℃冻存的组织，液氮充分研磨成粉末状，称取5~20 mg至1.5 mL离心管中，加入300 μL裂解液RTL和20 μL蛋白酶K（或者取适量组织于1.5 mL离心管中，加入裂解液RTL和蛋白酶K后，使用匀浆器匀浆），立即涡旋混匀，室温静置5分钟，10,000×g离心5分钟后取上清于新的1.5 mL离心管。
注意：组织量不要超过20 mg，否则可能导致RNA得率和质量下降。对于富含DNA的样本，如脾脏，建议使用5 mg。
(2) 细胞样本处理
按以下方式收集好细胞，在细胞中加入300 μL裂解液RTL和20 μL蛋白酶K，立即涡旋混匀1分钟，室温静置5分钟。
- 悬浮细胞的收集（收集细胞数量不要超过6× 10⁵）：估计细胞数量，300×g离心5分钟，将细胞收集到离心管中，小心吸弃所有培养基上清。
- 胰蛋白酶处理法：确定细胞数量，吸弃培养基，用PBS溶液洗涤细胞，吸弃PBS溶液，向细胞中加入含有0.10~0.25%胰蛋白酶的PBS溶液处理细胞，当细胞脱离容器壁时，加入含有血清的培养基，使胰蛋白酶失活，将细胞溶液转移至无菌无核酸酶的离心管中，300×g离心5分钟，收集细胞沉淀，小心吸弃所有上清。
注意：收集细胞时一定要将细胞培养液去除干净，否则会导致裂解不完全，影响RNA与磁珠的结合，造成RNA的产量下降。
4.2 加入600 μL裂解结合液BLB、20 µL磁珠MXVB，振荡或涡旋7~10分钟使磁珠吸附核酸。
4.3 转移至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.4 加入700 µL洗涤液DW1，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.5 加入700 µL洗涤液DW2，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.6 掌上离心机瞬时离心后，转至磁力架上吸附至溶液澄清，充分吸弃所有溶液，打开管盖，空气干燥3~5分钟。
注意：乙醇残留会抑制后续的酶反应，所以晾干时要确保乙醇挥发干净。切勿干燥过久，以免影响后续洗脱效果。
4.7 加入70 μL DNA酶反应液和10 μL DNase I，涡旋混匀，室温放置10~15分钟（每3~4分钟涡旋1次，每次30秒）。
4.8 涡旋混匀30秒，加入240 μL结合液RB，振荡或涡旋5分钟。
4.9 转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.10 加入700 µL洗涤液DW2，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.11 再次加入700 µL洗涤液DW2，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.12 掌上离心机瞬时离心后，转至磁力架上吸附至溶液澄清，充分吸弃所有溶液，打开管盖，空气干燥3~5分钟。
注意：乙醇残留会抑制后续的酶反应，所以晾干时要确保乙醇挥发干净。切勿干燥过久，以免影响后续洗脱效果。
4.13 加入50~100 µL洗脱液RE，高速涡旋2~3分钟打散磁珠。
4.14 45℃温浴5分钟，然后高速涡旋60秒。
4.15 瞬时离心收集管盖液滴至管中，转移至磁力架上进行磁分离。
4.16 把提取的RNA溶液转移至新的1.5 mL无菌无核酸酶的离心管中，如果不立即使用，请存储于-15~-25℃，长期保存请放置于-70℃或更低的温度。
方法的局限性
1. 对于某些取样不佳的样本，提取产物中可能存在少量杂质抑制PCR扩增。
2. 样本核酸的提取效果与样本收集、处理、运输和保存质量密切相关，其中任何失误都将会导致结果不准确。
This kit is designed for the isolation and purification of high-quality total RNA from various tissues and cells. The kit is based on superparamagnetic particle purification technology, eliminating the need for time-consuming alcohol precipitation. The resulting RNA can be used directly in various downstream applications such as RT-PCR, Real-Time RT-PCR, microarray analysis, Northern Blot, Dot Blot, Poly A selection, in vitro translation, RNase protection assays, and molecular cloning. The processed products are intended for scientific research and testing purposes only.
Storage Conditions
M1521688A, M1521688C, M1521688E, M1521688F, M1521688G, M1521688J: Store at room temperature, stable for 12 months.
M1521688B, M1521688D, M1521688I: Store at 2–8°C, stable for 12 months.
M1521688H: Store at -20°C, stable for 12 months.
Compatible Instruments
Manual extraction.
Precautions
Read this manual carefully before use.
Several buffers in this kit contain irritating guanidinium salts. Always wear gloves and handle according to standard safety precautions. Avoid contact of buffers with skin, eyes, and mucous membranes. In case of contact, flush immediately with plenty of water and seek medical attention.
If precipitation occurs in the solution due to low ambient temperatures, incubate at 30°C in a water bath until the precipitate is completely dissolved before use.
If the magnetic beads cannot be resuspended after vigorous shaking due to low ambient temperatures, do not use the beads.
Several buffers in this kit contain guanidinium salts. Do not treat with oxidizing disinfectants such as sodium hypochlorite, as this may release toxic gases. Dispose of waste according to medical waste regulations.
This kit components are designed to be used together as a system. Components from different lot numbers should not be interchanged or mixed.
Instructions for Use
1. Sample Requirements
Applicable sample types: Animal tissues, 4–6 × 10⁵ cells, and similar samples.
Sample storage and transport: Samples may be processed immediately or stored at -70°C or lower for up to 6 months. Avoid repeated freeze-thaw cycles. Transport samples using cold chain logistics.
Freeze-thaw requirements: Rapid freezing and rapid thawing are recommended; avoid repeated freeze-thaw cycles.
2. Equipment and Materials to be Supplied by User
Constant temperature water bath (for use when precipitation occurs in solutions)
Dry block heater
Magnetic separation rack (for 1.5/2.0 mL tubes)
1.5/2.0 mL sterile, nuclease-free centrifuge tubes
Pipettes and tips of various volumes (10, 20, 200, 1000 μL)
3. Reagent Preparation
Check solutions for any precipitation and confirm that the magnetic beads can be resuspended.
4. Operating Procedure
Recommendation for magnetic separation (starting from step 4.3): After placing the centrifuge tube on the magnetic rack, gently rotate it left and right. Once the magnetic beads have aggregated against the tube wall closest to the magnet, gently invert the entire rack several times to collect any beads from the cap onto the tube wall. Allow to stand for several minutes until the solution becomes clear (standing time depends on the strength of the magnetic rack).
4.1 Sample Pre-treatment
(1) Tissue Sample Processing
Take fresh or -80°C frozen tissue and grind thoroughly into a fine powder using liquid nitrogen. Weigh 5–20 mg of the powder and transfer it to a 1.5 mL centrifuge tube. Add 300 μL Lysis Buffer RTL and 20 μL Proteinase K (alternatively, place an appropriate amount of tissue into a 1.5 mL centrifuge tube, add Lysis Buffer RTL and Proteinase K, and homogenize using a homogenizer). Immediately mix by vortexing and let stand at room temperature for 5 minutes. Centrifuge at 10,000 × g for 5 minutes, then transfer the supernatant to a new 1.5 mL centrifuge tube.
*Note: Do not use more than 20 mg of tissue, as this may reduce RNA yield and quality. For DNA-rich samples such as spleen, 5 mg is recommended.*
(2) Cell Sample Processing
Collect cells as described below, then add 300 μL Lysis Buffer RTL and 20 μL Proteinase K to the cell pellet. Immediately vortex for 1 minute and let stand at room temperature for 5 minutes.
Collection of suspension cells (do not exceed 6 × 10⁵ cells): Estimate the cell count. Centrifuge at 300 × g for 5 minutes to pellet the cells in a centrifuge tube. Carefully aspirate and discard all culture medium supernatant.
Trypsinization method: Determine the cell count. Aspirate the culture medium and wash the cells with PBS solution. Aspirate the PBS and add PBS solution containing 0.10–0.25% trypsin to detach the cells. When the cells detach from the vessel surface, add serum-containing medium to inactivate the trypsin. Transfer the cell suspension to a sterile, nuclease-free centrifuge tube and centrifuge at 300 × g for 5 minutes to pellet the cells. Carefully aspirate and discard all supernatant.
Note: It is critical to completely remove the cell culture medium during cell collection. Residual medium can lead to incomplete lysis, affecting RNA binding to the magnetic beads and resulting in reduced RNA yield.
4.2 Add 600 μL Lysis/Binding Buffer BLB and 20 μL Magnetic Beads MXVB. Shake or vortex for 7–10 minutes to allow nucleic acid binding to the beads.
4.3 Place the tube on the magnetic rack for magnetic separation until the solution is clear, then aspirate and discard the supernatant.
4.4 Add 700 μL Wash Buffer DW1. Vortex for 30 seconds, then place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.5 Add 700 μL Wash Buffer DW2. Vortex for 30 seconds, then place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.6 Perform a quick spin using a mini centrifuge, then place the tube back on the magnetic rack and allow beads to collect until the solution is clear. Carefully aspirate and discard all residual liquid. Open the tube cap and air-dry the beads for 3–5 minutes.
Note: Residual ethanol will inhibit downstream enzymatic reactions. Ensure ethanol has completely evaporated during drying. Do not over-dry the beads, as this may affect subsequent elution efficiency.
4.7 Add 70 μL DNase Reaction Buffer and 10 μL DNase I. Mix by vortexing and incubate at room temperature for 10–15 minutes (vortex briefly every 3–4 minutes, 30 seconds each time).
4.8 Vortex for 30 seconds, then add 240 μL Binding Buffer RB. Shake or vortex for 5 minutes.
4.9 Place the tube on the magnetic rack for magnetic separation until the solution is clear, then aspirate and discard the supernatant.
4.10 Add 700 μL Wash Buffer DW2. Vortex for 30 seconds, then place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.11 Add another 700 μL Wash Buffer DW2. Vortex for 30 seconds, then place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.12 Perform a quick spin using a mini centrifuge, then place the tube back on the magnetic rack and allow beads to collect until the solution is clear. Carefully aspirate and discard all residual liquid. Open the tube cap and air-dry the beads for 3–5 minutes.
Note: Residual ethanol will inhibit downstream enzymatic reactions. Ensure ethanol has completely evaporated during drying. Do not over-dry the beads, as this may affect subsequent elution efficiency.
4.13 Add 50–100 μL Elution Buffer RE and vortex at high speed for 2–3 minutes to fully disperse the beads.
4.14 Incubate at 45°C for 5 minutes, then vortex at high speed for 60 seconds.
4.15 Briefly centrifuge to collect any droplets from the cap into the tube, then place on the magnetic rack for magnetic separation.
4.16 Transfer the extracted RNA solution to a new 1.5 mL sterile, nuclease-free centrifuge tube. If not used immediately, store at -15 to -25°C. For long-term storage, place at -70°C or lower.
Limitations of the Method
For certain poorly collected samples, trace impurities in the extracted product may inhibit PCR amplification.
The efficiency of nucleic acid extraction is closely related to the quality of sample collection, handling, transport, and storage. Any error in these processes may lead to inaccurate results.
ZJ26F0535378
危险品化学品经营许可证（带存储）