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View additional product information for PureLink™ RNA Mini Kit - FAQs (A29839, 12183026, 12183025, 12183020, 12183018A)
11 product FAQs found
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.
We recommend using 1 volume of ethanol as stated on page 19 of the PureLink RNA Mini Kit (Cat. Nos. 1283018A, 12183020, 12183025) User Guide, however, 1.5 volumes of ethanol as stated in the Reference Guide will work as well.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation 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.