Invitrogen™ PureLink™ RNA Mini Kit

Product Code.	Quantity	Product Type	unitSize
10359103	10 Preps	RNA Mini Kit	Each
10307963	50 Preps	RNA Mini Kit	Each
13355364	250 Preps	RNA Mini Kit	250 preps
15914204	50 Cartridges	RNA Mini Columns	Pack of 50
10043742	50 Cartridges	Homogenizer	50 reactions

Product Code. 10359103 Supplier Invitrogen™ Supplier No. 12183020

Description

The PureLink RNA Mini Kit is a column-based kit used to isolate high-quality total RNA from a wide variety of sample types in 20 minutes using standard laboratory equipment.

The PureLink RNA Mini Kit is a column-based kit used to isolate high-quality total RNA from a wide variety of sample types in 20 minutes using standard laboratory equipment. The kit includes RNase-free lysis and wash solutions that protect RNA from RNases while liberating the RNA from DNA, proteins, and other cellular debris. The fast spin-column workflow is ideal for processing low to mid-throughput batch sizes. The advanced PureLink RNA Mini spin column design allows for maximum sample input (200 mg of tissue) and RNA recovery (up to 1000 μg). This means that users can process both small and large sample sizes with the same RNA isolation kit.

• Process sample inputs from 5–200 mg of animal/plant tissue or from 0.5 x 10⁶ to 10⁸ tissue culture cells with one spin column in 20 minutes
• Isolate more RNA than competitor mini spin columns: up to 1000 μg of total RNA from one column
• Use isolated RNA in most applications without the need for further DNase treatment (very low minimal residual DNA carry over)
• Flexible–optional on-column or post-isolation DNase treatment is available with PureLink DNase Set (Cat. No. 12185010) for easy removal of residual DNA

Extra-large binding capacity enables rapid RNA purification using standard laboratory equipment

The PureLink RNA Mini Kit provides rapid purification of total RNA from a wide range of cells and tissue types to yield up to 1000 μg of purified RNA from a single extraction (see Figure 1). High-quality total RNA can be obtained from mini- to midi-prep amounts of starting material with just trace amounts of residual genomic DNA contamination. The extra-large binding capacity enables one kit to handle most RNA isolation needs in a quick 20 minute protocol without the need for special sample processing instruments.

Easy, optional, on-column DNase treatment for sensitive applications

The PureLink RNA Mini Kit columns are highly efficient for isolating high quality total RNA while removing the majority of genomic DNA. In general, most applications requiring RNA from animal tissue or mammalian cell lines do not require additional DNase treatment. However, some applications such as gene expression analysis by qRT-PCR without intron-spanning primers or working with samples from organisms with very small or no introns may require more complete removal of residual contaminating DNA. The PureLink DNase Set (12185010) allows for convenient on-column digestion of DNA during the RNA isolation protocol. Treating with DNase while 'on-column' is easier and allows higher RNA recovery than treating with DNase after the RNA has been isolated. The PureLink DNase Set can also be used to remove residual DNA from RNA that has been previously purified. Both the 'on-column' and post-RNA purification workflows are options available with the PureLink DNase Set.

Simplified, non-toxic RNA purification

The PureLink RNA Mini Kit utilizes non-toxic guanidine-isothiocyanate lysis buffer to protect the RNA during the isolation steps. Special RNase-free reagents combined with certified RNase-free silica membranes in a unique column configuration allow for a safe and easy procedure that can typically be completed in less than 20 minutes without the need of hazardous phenol/chloroform extraction, CsCl centrifugation, or LiCl or alcohol precipitation.

The PureLink RNA Mini Kit is recommended for use with the Homogenizer (Cat. No. 12183026), designed to homogenize cell or tissue lysates via centrifugation, prior to nucleic acid purification. The Homogenizer is especially effective for clarifying particulates from plant tissues.

Specifications

• Content And Storage: • 20 mL Lysis Buffer; store at room temperature
  • 10 mL Wash Buffer I; room temperature
  • 4 mL Wash Buffer II; room temperature
  • 3 mL RNase-free Water; room temperature
  • 10 PureLink Spin Cartridges with Collection Tubes; room temperature
  • 10 Collection Tubes; room temperature
  • 10 Recovery Tubes; room temperature
• Isolation Technology: Silica Spin Column
• Purification Time: 20 min. (After sample homogenization)
• Elution Volume: 30–300 μL
• Sample Type: Bacteria, Blood, Cells, Plant, Tissue, Yeast, Enzymatic Reactions
• Final Product Type: Total RNA
• High-throughput Compatibility: Not High-throughput Compatible (Manual)
• For Use With (Application): RT-PCR, qPCR, cDNA Library Construction, NGS, Microarray Analysis, Blot Hybridization, Northern Blotting, In Vitro Translation, Nuclease Protection Assays, Nucleic Acid Labeling
• Product Type: RNA Mini Kit
• Quantity: 10 Preps
• Scale: Mini
• Shipping Condition: Room Temperature
• Starting Material Amount: Bacteria: ≤10⁹ cells, Blood: ≤0.2 mL, Cells: ≤5 x 10⁷, Plant: ≤250 mg, Tissue: ≤200 mg, Yeast: ≤5 x 10⁸ cells, Enzymatic reactions: ≤1.2 mL
• Yield: Up to 350 μg

Frequently Asked Questions (FAQs)

I've isolated RNA using the PureLink RNA Mini Kit, but see inhibition of downstream enzymatic reactions. What could be causing this?

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.

I'm seeing RNA degradation after RNA extraction using the PureLink RNA Mini Kit. What happened?

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.

I'm getting low RNA yield when using the PureLink RNA Mini Kit. What could be the cause of this and what do you suggest I try?

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.

I'm using the PureLink RNA Mini Kit and my RNA spin cartridge is clogged. What should I do?

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.

What are the differences in technology between the PureLink RNA Mini Kit and RiboPure RNA Purification Kits?

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.

Do you have any data showing differences in RNA extraction with TRIzol Reagent, the PureLink RNA Mini Kit, ChargeSwitch Total RNA Cell Kit or the TRIzol Plus RNA Purification Kit?

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.

I am using the PureLink RNA Mini Kit, but want to perform an on-column DNase I digestion. Do you have suggestions for this?

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.

I am using your PureLink RNA Mini Kit. Can I isolate small RNA using this kit?

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.

The PureLink RNA Mini Kit (Cat. Nos. 1283018A, 12183020, 12183025) protocol for purifying RNA from animal and plant cells in the user guide differs from the quick Reference for the volume of ethanol. Which one is correct?

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.

Are the collection tubes for the PureLink RNA Mini Kit (Cat. No. 12183018A) available as a stand-alone item?

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.

Do you offer a kit that will allow me to sequentially isolate gDNA and total RNA from my tissue sample that is not an FFPE (formalin-fixed, paraffin-embedded) sample?

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.

Safety and Handling

Product Identifier

• PureLink RNA Mini Kit

Signal Word

• Danger

Hazard Category

• Acute toxicity Category 4
• LONG-TERM AQUATIC HAZARD Chronic 3
• Serious eye damage/eye irritation Category 1
• Serious eye damage/eye irritation Category 2
• Skin corrosion/irritation Category 1 C
• Skin corrosion/irritation Category 2

Hazard Statement

• H302-Harmful if swallowed.
• H314-Causes severe skin burns and eye damage.
• H315-Causes skin irritation.
• H319-Causes serious eye irritation.
• H412-Harmful to aquatic life with long lasting effects.

Precautionary Statement

• P260-Do not breathe dust/fume/gas/mist/vapours/spray.
• P264-Wash hands thoroughly after handling.
• P270-Do not eat, drink or smoke when using this product.
• P273-Avoid release to the environment.
• P280-Wear protective gloves/protective clothing/eye protection/face protection.
• P301+P310-IF SWALLOWED: Immediately call a POISON CENTER/doctor /
• P302+P352-IF ON SKIN: Wash with plenty of water/soap.
• P303+P361+P353-IF ON SKIN (or hair): Take off immediately all contaminated clothing. Rinse skin with water/ shower.
• P305+P351+P338-IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
• P310-Immediately call a POISON CENTER/doctor/.
• P330-Rinse mouth.
• P332+P313-If skin irritation occurs: Get medical advice/attention.
• P337+P313-If eye irritation persists: Get medical advice/attention.
• P362+P364-Take off contaminated clothing and wash it before reuse.
• P501b-Dispose of contents/container in accordance with local/regional/national/international regulations.

Supplemental information

• MIXTURE LIST-Contain: Thiocyanic acid

For Research Use Only. Not for use in diagnostic procedures.