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View additional product information for PureLink™ RNA Mini Kit - FAQs (A29839, 12183026, 12183025, 12183020, 12183018A)
21 product FAQs found
若纯化RNA中存在乙醇或盐，会抑制下游酶反应。应确保试剂盒中洗涤缓冲液的使用顺序正确，并在洗脱前去除洗涤缓冲液II的流穿液。将纯化柱放置到洗涤管中并以最大转速离心2-3分钟，从而使纯化柱完全干燥。
RNA可能被RNase污染。应确保使用了无RNase的设备，并经常更换手套。不恰当的处理也会导致RNA降解。应确保立即处理样品，并且在加入裂解缓冲液后快速进行裂解。最后，富含RNase的组织（如大鼠胰腺）可能需要加入RNase抑制剂或灭活剂以防止RNA降解，或需要加入更多的裂解缓冲液。
低RNA得率可能是由于以下原因：
•裂解和匀浆不完全：应确保在每毫升裂解液中加入10 µL的2-巯基乙醇、所有步骤在室温下进行、减少起始样品量、使用合适的匀浆方法和/或将组织样品切成小块，以保证组织完全浸没于裂解液中。
•起始样品质量差：使用新鲜的样品，收集样品后立即处理。
•可能未在洗涤缓冲液II中加入乙醇。
•可能使用了不正确的洗脱条件：在离心前加入无RNase水并孵育1分钟，遵循使用手册中的洗脱建议。您也可采取二次洗脱步骤，以回收更多RNA。
匀浆不完全或加入乙醇后发生沉淀分散，会导致RNA纯化柱堵塞。可通过离心净化匀浆液，除去所有颗粒物或粘稠物。在匀浆液中加入乙醇后要使形成的所有沉淀完全分散。仅将上清液上样到RNA纯化柱上，以避免堵塞。
PureLink RNA小量提取试剂盒提供快速的总RNA柱纯化，无需有机溶解（苯酚/氯仿）。单次提取可获得多达1,000 µg的纯化RNA。RiboPure RNA纯化试剂盒结合了苯酚/硫氰酸胍溶液和玻璃纤维过滤纯化法。
我们的网站（https://www.thermofisher.com/content/dam/LifeTech/migration/en/images/ics-organized/applications/nucleic-acid-purification/data-image/560-wide.par.83692.image.559.294.1.gif）中有图表对比了上述试剂盒在纯度检测和RNA完整指数（RIN）方面的差异。
可使用PureLink DNase（货号12185010）进行柱上消化。请查看手册附录第63页的实验方案。
PureLink RNA小量提取试剂盒仅经过验证能够回收> 200 nt的RNA。但是，您可尝试以下改进方法，从而分离包括小RNA在内的总RNA：
•在纯化柱上样之前，向裂解物中加入更多乙醇。乙醇终浓度应至少为55%。
•将洗液1的乙醇浓度增加至最低55%。
•不用柱上DNase处理以减少对小RNA的损失。
对于细菌样品，您可使用我们的TRIzol Max细菌RNA分离试剂盒（货号16096040），该试剂盒在匀浆时含有额外的试剂，有助于细菌细胞壁的裂解。您也可以使用我们的PureLinkRNA小量提取试剂盒（货号12183018A），使用该试剂盒时，需要准备溶菌酶溶液以溶解细胞壁。
The presence of ethanol or salt in the purified RNA can inhibit downstream enzymatic reactions. Ensure that you are using the correct order of wash buffers in the kit for washing, and that Wash Buffer II is discarded in the flow-through. Place the spin cartridge into the wash tube and centrifuge the spin cartridge at maximum speed for 2-3 minutes to completely dry the cartridge.
The RNA could have been contaminated with RNase. Ensure that you are using RNase-free equipment and change gloves frequently. Improper handling can also result in RNA degradation. Ensure samples are processed immediately, and that the lysis is performed quickly after adding the lysis buffer. Lastly, tissues rich in RNase (such as rat pancreas) may require the addition of RNase inhibitors or inactivators to protect the RNA from degradation, or a larger volume of lysis buffer.
Low RNA yield can occur due to the following:
- Incomplete lysis and homogenization: ensure that 10 µL of 2-mercaptoethanol was added per milliliter of lysis buffer, perform all steps at room temperature, decrease the amount of starting material used, use proper homogenization methods, and/or cut tissue samples into smaller pieces to ensure complete tissue immersion in the lysis buffer.
- Poor quality starting material: use fresh samples and process immediately after collection.
- Ethanol may not have been added to Wash Buffer II.
- Incorrect elution conditions may have been used: Add RNase-free water and incubate for 1 minute before centrifugation, following the recommendations for elution in the manual. You can also perform a second elution step to recover more RNA.
Incomplete homogenization or dispersal of precipitate after ethanol addition can lead to clogging of the RNA spin cartridge. Clear the homogenate and remove any particulate or viscous material by centrifugation. Completely disperse any precipitate that forms after adding ethanol to the homogenate. Load only the supernatant onto the RNA spin cartridge to avoid clogging.
The PureLink RNA Mini Kit provides rapid column-based purification of total RNA, without organic lysis (phenol/chloroform). You can obtain up to 1,000 µg of purified RNA from a single extraction. The RiboPure RNA Purification Kits combine a phenol/guanidine thiocyanate solution with a glass-fiber filter purification method.
Please visit our website (http://www.thermofisher.com/content/dam/LifeTech/migration/en/images/ics-organized/applications/nucleic-acid-purification/data-image/560-wide.par.83692.image.559.294.1.gif) for a graph showing purity measurements and RNA integrity number (RIN) comparison of the abovementioned kits.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
For on-column digestion, PureLink DNase (Cat. No. 12185010) can be used. Please see the protocol in the appendix of the manual on page 63.
The PureLink RNA Mini Kit was only validated to recover RNA greater than 200 nt. However, you can try the following modifications to isolate total RNA including small RNA:
- Add more ethanol to the lysate before loading it onto the column. The final ethanol concentration should be at least 55%.
- Increase the ethanol concentration in Wash 1 to at least 55%.
- No on-column DNase treatment can be used without loss of the small RNA.
For bacterial samples, you could use our TRIzol Max Bacterial RNA Isolation Kit (Cat. No. 16096040), that contains an extra reagent during homogenization to help with lysis of the bacterial cell walls. You could also use our PureLink RNA Mini Kit (Cat. No. 12183018A), for which you would need to prepare a lyosyme solution to lyse the cell wall.
These kits have not been tested. However, these kits should work with the TaqMan Advanced miRNA Assays. We recommend checking the quality and quantity of small RNAs post-isolation by NanoDrop and Bioanalyzer.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
Yes, the collection tubes for the PureLink RNA Mini Kit (Cat. No. 12183018A) are available separately as Cat. No. 12282-100.
For additional spin cartridges, go through catalog number A29839. However, that one is not listed on the website but it can be ordered by Quick Order.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
We offer TRIzol reagent that will allow isolation of DNA and RNA from the same sample. Alternatively, we have the following method that has been validated by our R&D team; for sequential isolation of gDNA and total RNA from the same sample. This method involves using 2 of our kits: 1) PureLink RNA Mini Kit (Cat. No. 12183018A, 12183020, 12183025) and 2) PureLink Genomic DNA Mini Kit (Cat. No. K182002, K182000, K182001).
The protocol is detailed below:
Before starting:
- Label all spin columns and buffers from each kit with kit names to prevent confusion.
- Prepare lysis buffer and wash buffers according to the protocol from each kit.
1. Preparing lysates:
- Add 300 µL of lysis buffer (from Purelink RNA Mini Kit, beta-mercaptoethanol added) to cell or tissue sample, lyse the cells as recommended.
2. DNA isolation:
- Load all of the lysate directly onto a Purelink gDNA column (from PureLink Genomic DNA Mini Kit), save flow-through for RNA isolation.
- Centrifuge the Purelink gDNA column at 10,000 x g for 1 min.
- Wash the Purelink gDNA column with 500 µL of Wash Buffer 1 (from PureLink Genomic DNA Mini Kit, ethanol added), centrifuge at 10,000 x g for 1 min.
- Add 500 µL of Wash Buffer 2 (from PureLink Genomic DNA Mini Kit, ethanol added), centrifuge at maximum speed for 3 min to dry the membrane.
- Add 100 µL of Elution Buffer (from PureLink Genomic DNA Mini Kit), incubate at room temperature for 1 min and centrifuge at 10,000 x g for 1 min (yield can be increased if an optional second elution step is added).
- This is purified gDNA.
3. RNA isolation:
- To the above saved flow-through, add same volume of 70% ethanol, mix well and load the lysate/ethanol mix (including all precipitates) onto an RNA spin cartridge (from Purelink RNA Mini Kit).
- Centrifuge at 12,000 x g for 15 sec. Discard flow-through.
- Wash the RNA spin cartridge with 700 µL of Wash Buffer 1 (from Purelink RNA Mini Kit, ethanol added), centrifuge at 12,000 x g for 15 sec.
- Wash twice with 500 µL of Wash Buffer 2 (from Purelink RNA Mini Kit, ethanol added). After the second wash, centrifuge at 12,000 x g for 1 min to dry the membrane.
- Add 50 µL of RNase-free water onto the RNA spin cartridge, incubate at room temperature for 1 min and centrifuge at 12,000 x g for 2 min (yield can be increased if an optional second elution step is added).
- This is purified RNA.