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Specialized RNA Isolation (mRNA, miRNA, viral RNA)

Please use the selection guide to view the kits we offer for mRNA purification, as well as the differences between them. Read more about our Dynabeads® mRNA kits in the Dynabeads® webpage.

RNAs less than 200 nt are lost using the columns in the RNAqeous® family of kits. These kits are not suitable for isolation of 5S rRNA or tRNA. The RNAqueous®-Micro Total RNA Isolation Kit does, however, offer a modified protocol to allow isolation of miRNA from the sample.

  • Precipitation of miRNA: Follow the instructions for the mirVANA™ miRNA Isolation Kit or the mirVANA™ PARIS™ RNA and Native Protein Purification Kit.
  • Double phenol/guanidium (chaotropic reagent) extraction: Disrupt tissue with phenol/guanidine, then extract with phenol, centrifuge, remove the supernatant, and precipitate the RNA.
  • Glass-fiber filter: Disrupt the tissue in a chaotropic agent, bind the RNA to a glass-fiber filter, then wash off unbound DNA/protein/salt/nucleotides and elute the RNA.

For recovery of miRNA of formalin-fixed samples, we recommend using RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE. You can isolate total and miRNA using the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE, then use that prep for enrichment of miRNA using the enrichment protocol described in the instructions for the miRVana™ kit. For unfixed LCM samples, you could use an RNAqueous® kit.

No, unfortunately, small RNAs will be lost using this purification method due to the low molecular weight cutoff from the column (~200 bp).

Yes, using a modification of the Megaplex™ Pools for microRNA Expression Analysis Protocol (P/N 4399721 Rev. B). In this protocol, scale the reverse transcription and preamplification reactions to include all of the cell lysate.

Yes. You will need to follow the alternate protocol, available in the following link.

The Poly(A)Purist™ Kit and MicroPoly(A)Purist™ Kit use oligo(dT) cellulose-based selection, while the Poly(A)Purist™ MAG kit uses oligo(dT) magnetic bead-based purification. The Poly(A)Purist™ Kit and Poly(A)Purist™ MAG Kit are used with total RNA preparations, while the MicroPoly(A)Purist™ Kit can isolate mRNA from a total RNA preparation or from small amounts of tissue or mammalian cells directly.

Yes, you can add a tenth volume of 3 M NaOAc before doing an ethanol precipitation.

With regard to species specificity, the following organisms have 0 mismatches for the LSU/SSU probes (LSU (28S) / SSU (18S) Probe perfect matches). This means the probes will remove the 18S and 28S sequences from the following species.

Species from the Land

Species from the Sea

Plants

Human, mouse, rat, frog, rabbit, cow, pig, chicken, Drosophila,* Caenorhabditis elegans, midge, mosquito, yeast (Saccharomyces cerevisiae, Schizosaccharomyces pombe)

Sea squirt, coelacanth, lancelet, eel, salmon, sturgeon, ratfish, lamprey, brown plankton, zooplankton

Arabidopsis thaliana, Brassica napus, rice, tomato, Humulus lupulus, Zea mays, wheat, soybean, pine, aspen

* The 28S and 18S probes in this kit respectively align 100% to the Drosophila 18S and 28S rRNA sequence. However, there are reports suggesting Drosophila (insect) 28S rRNA split into 2 smaller fragments co-migrating with 18S. The exact split site is not identified yet. But based on customer feedback, this kit does not remove the split 28S rRNA completely. We have not tested Drosophila samples with this kit.
The 5S and 5.8S sequences are more divergent, and thus the best probes possible have reduced species breadth:
5S: zero to 1 mismatch: human, mouse, rat, frog, chicken, Drosophila, C. elegans, A. thaliana, Z. mays
5.8S: zero mismatches: human, mouse, rat

The RNA is easily quantitated using UV absorbance at 260 nm or a Quant-iT™ RNA Assay Kit. To verify rRNA depletion, electrophorese your sample on an agarose gel or use an Agilent® 2100 Bioanalyzer. Agarose gel electrophoresis should show depletion of 18S and 28S rRNA bands compared to a control sample. Absence of contaminating DNA and RNA degradation may also be confirmed by agarose gel electrophoresis. The efficiency of rRNA depletion in the purified RNA purified, RNA degradation, and RNA concentration can also be analyzed using a bioanalyzer.

You would have to separate the virus from the cells prior to isolation to avoid contamination of the sample with cellular nucleic acids.

Here is a list of DNA/RNA Purification replacement buffers we offer as standalone.

Automated RNA Isolation

The MagMAX™ Express-96 plates, deep well plates (squared wells), and PCR plates (skirted) can be used with the instrument. Each plate type has an optimized MagMAX™ Express-96 head and tips. Of the three plates, the MagMAX™ Express-96 plate can be used with all three kinds of magnets. The volume of the deep well plates has to be a minimum of 2.2 mL (2.0 mL capped) with squared wells.

Yes. Both deep well plates and MagMAX™ Express-96 plates can be used during the same run. Therefore, it is possible to start the processing by using larger volumes (in a deep well plate) and elute the purified sample to a smaller volume (in a MagMAX™ Express96 plate).

We offer a complete MagMAX™ Express-96 system called the MagMAX™ Express-96 Magnetic Particle Processor for the purification and processing of proteins, DNA, RNA, and cells in a 96-well format. The processor handles particles automatically according to the preloaded purification protocols.

The MagMAX™ Express 96 magnets are made of material that is very stable. The magnetic field will not get weaker. However, extreme mechanical force or heating can cause damage to the magnets. Note: It is very important to keep the MagMAX™ Express-96 heads away from each other and other magnets at all times. Clashing of the magnets together can cause serious damage to the magnets.

The MagMAX™ Express-96 system should not be kept in close proximity to magnetic tapes, computer discs, or other magnetic storage systems, such as credit cards, as these can be damaged by the strong magnetic field of the MagMAX™ Express-96 heads. Do not hold the MagMAX™ Express-96 heads close to a PC display, since this may cause damage to the display. Do not use metal tools when handling MagMAX™ Express-96 heads.

Warning: This product contains very strong permanent magnets. People wearing a pacemaker or metallic prosthesis should not use this product. A pacemaker or prosthesis may be affected or damaged if it comes in close contact with a strong magnetic field.

The heating block is located inside the instrument and can be used automatically during the protocol. Both MagMAX™ Express-96 plates and PCR plates (skirted) can be heated using specially designed, interchangeable heating blocks. Any number of heating steps can be added to the protocol. During the protocol, when the protocol enters the heating step, the plate is automatically moved to the dedicated heating position for heating. After the heating step, the protocol continues automatically to the next step.

Note: No cooling steps can be added.

We strongly recommend that you keep the specified volumes within the defined limits to avoid spillover of the reagents.

There is a pause function if you hit "stop" in the middle of a run. If you hit start again, the run continues. If you hit stop twice, the run stops completely. There is no way to begin a run in the middle of a protocol.

Mixing with the tip comb is designed to occur below the liquid surface. This prevents aerosols and splashing from occurring when using the MagMAX™ Express instrument.

There are two rows of 12 magnetic heads each, and 2 plates for loading.

Deep well plates are recommended. Use a deep well configuration for larger sample inputs (up to 400 μL).

Yes, you can use 0.1 mM EDTA as an alternative (though beads may pellet slower).

This table shows the reagents shared among different MagMAX™ kits:

 

AM1835

AM1836

AM1939

AM1830

AM1837

AM1839

AM1840

Lysis/Binding Solution Conc.

X

X

X

X

X

X

X

RNA Binding Beads

X

X

X

X

X

X

X

Wash Soln. 1 Conc.

X

X

X

X

X

X

X

Wash Soln. 2 Conc.

X

X

X

X

X

X

X

Elution Buffer

X

X

X

X

X

X

X

Lysis/Binding Enhancer

 

X

X

X

X

X

X

Carrier RNA

X

X

X

 

 

 

X

TURBO™ DNase

 

 

 

X

X

X

 

MagMAX™ TURBO™  DNase Buffer

 

 

 

X

X

X

 

Zirconia Bead Tubes

 

 

 

 

 

 

X

TRI Reagent®

 

 

 

 

 

X

 

RNA Rebinding Conc. or Bead Resusp. Soln.

X

 

 

x

 

 

 

*Fill volumes may differ from kit to kit; however, the same composition is used.

The MagMAX™ Magnetic Particle Processor can capture small RNAs if you adjust the binding solution and Wash Solution 1 to 50% isopropranol. The Wash Solution 2 and Elution Solution remain the same. You can expect highest miRNA recovery with the modified protocol for Cat. No. AM1839.

Yes, carrier RNA can be ordered separately. The Cat. No. is 4382878.

Yes, adjust the binding solution and wash solution 1 to 50% isopropanol to precipitate small RNAs. However, we have not yet tested yield with this protocol modification.

Yes, the Applied Biosystems™ MagMAX™ mirVana Total RNA Isolation Kit (Cat. No. A27828) will isolate both small and large RNAs from a variety of samples (serum, plasma, blood, tissue, cells, urine).

It is a personal preference as to which magnetic stand to purchase. Cat. No. AM10027 can be easier if using a pipettor for liquid handling, as beads clump to the side, Cat. No. AM10050 if using a robotic liquid handler, as beads clump in a ring.

Yes, the iPrep™ PureLink Total RNA Kit will do this. When prompted on screen, you could select the "No DNase" option. This will result in collection of all nucleic acids present in the sample. However, this kit was not optimized to purify DNA, so there would be preferential binding for RNA over DNA due to the GITC in the lysis/binding buffer.

The iPrep™ Purification Instrument allows for automated purification of nucleic acids from up to 13 samples (12 samples and 1 positive control). You will need to purchase an iPrep™ Kit, which contains the sample preparation reagents and reagents, as well as the iPrep™ Protocol Card, which is preprogrammed with the purification protocol that directs the volume of reagents used and incubation time.

iPrep™ Kit

Recommended iPrep™ Card

iPrep™ ChargeSwitch® gDNA Tissue Kit

iPrep™ gDNA Blood and Tissue Card

iPrep™ ChargeSwitch® Forensic Kit

iPrep™ Forensic Card (includes buccal protocol)

iPrep™ ChargeSwitch® Buccal Cell Kit

iPrep™ Forensic Card (includes buccal protocol)

There are 4 magnetic head options available for the KingFisher™ Flex Magnetic Particle Processor:

- KingFisher™ Flex head for MagMAX™ Express-96 Deep Well plate

- KingFisher™ Flex head for KingFisher™ 24 Deep Well plate<br/>

- KingFisher™ Flex head for KingFisher™ 96 plate<br/>

- KingFisher™ Flex head for PCR plate, skirted

No, the 96 well standard and 96 Deep Well heads are not interchangeable. However, the same tip combs can be used for both instruments.

The consumables for the KingFisher™ Flex instrument with the 96 Deep Well head are the same as for the MagMAX™ Express-96 instrument. They are listed below: 

- MagMAX™ Express-96 Deep Well Plates (Cat. No. 4388476)

- MagMAX™ Express-96 Standard Plates (Cat. No. 4388475)

- MagMAX™ Express-96 Deep Well Tip Combs (Cat. No. 4388487)

- MagMAX™ Express-96 Standard Tip Combs (Cat. No. 4388488)

- MicroAmp™ Clear Adhesive Film (optional): (Cat. No. 4306311)

For the KingFisher™ Flex instrument with the 24 Deep Well head, the following consumables are required:

- 24 Deep-Well Plates for KingFisher™ Flex: (Cat. No. 95040480)

- 24 Deep Well Tip Combs for KingFisher™ Flex: (Cat. No. 97002610)

No, we strongly recommend that you use the MagMAX™ plates or KingFisher™ plates since they were specifically designed to be used with KingFisher™ Flex tip combs to attain maximal performance. Plates from other manufacturers may not be compatible with the KingFisher™ Flex heating blocks. They may also cause unexpected problems, such as cross-contamination, due to the divergent well volume and bottom height of the plate.

Yes, reaction volumes can be scaled up, and all other volumes (i.e. wash volumes) will have to be increased by the same fold difference. It is generally not recommended to scale up reaction volumes more than 2-fold unless the plate's volume capacity will allow it.

Yes, this can be done by sample splitting. Set the protocol so that the magnet will collect samples from multiple wells before continuing to the next step.  Be sure not to overload.

For nucleic acid extraction, we recommend using our MagMAX™ kits and MagJET kits. Other magnetic beads should work well as long as they are 0.5 to 10 µm in size. However, you will have to write your own KingFisher™ protocols using the Thermo Scientific™ BindIt™ software and validate/optimize the protocol on your own.

BindIt™ software is compatible with Windows™ XP (Professional with Service Pack 3), Windows™ Vista (Service Pack 2), Windows™ 7 (Service Pack 1), and Windows™ 8.

No, manufactured protocols are locked for editing, but they can be renamed using the “save as” function and edited as a new version.

Yes, the MagMAX™ Express-96 instrument can connect to a computer with the most recent BindIt™ software v3.3.1.7 installed.  A serial port to USB converter is required to connect.  Existing .kf2 files will not be editable in the BindIt™ software but it is possible to re-write a .kf2 file as a .bdz file so that it is editable. Please see this application note for detailed instructions on how to do that.

Protocols cannot be transferred back from the KingFisher™ Flex instrument to the computer.  Protocols need to be previously stored in the BindIt™ software to make edits.

KingFisher™ Flex uses a USB port, whereas the MagMAX™ Express- 96 requires a RS-232 port.

The KingFisher™ Flex magnets are made of material that is very stable. The magnetic field will not get weaker. However, extreme mechanical force or heating can cause damage to the magnets.

Caution: It is very important to keep the KingFisher™ Flex heads away from each other and from other magnets at all times. Clashing together of the magnets may cause serious damage to the magnets.

The KingFisher™ Flex magnets are made of material that is very stable. The magnetic field will not get weaker. However, extreme mechanical force or heating can cause damage to the magnets.

Caution: It is very important to keep the KingFisher™ Flex heads away from each other and from other magnets at all times. Clashing together of the magnets may cause serious damage to the magnets.

This can be done by running the Change magnet protocol under the Maintenance menu using the “up” and “down” cursor keys.

Both deep-well plates and KingFisher™ 96 plates can be used during the same run. Therefore, it is possible to start the processing by using larger volumes (in a deep-well plate) and elute the purified sample to a smaller volume (in a KingFisher™ 96 or MagMAX™ 96 plate).

The heating block warms up at the rate of about 10 degrees C per minute, so it will take about 6 minutes.

We strongly recommend that you maintain the specified volumes within the defined limits to avoid spillover and to help ensure the best performance at the most efficient level.

The transport locks are only necessary when relocating the instrument.

Set up the protocol on the BindIt™ software. After the first 8 plates are done, the instrument will pause and instruct you to switch out the plates.

No, the processing volume refers to the maximum volume that can be held in the 24 Deep Well plates. This incorporates the volume of binding buffers and beads, so when scaling up reaction volumes, it is important to make sure that the total volume does not exceed 5 mL.

Yes, it comes with one processing plate, two elution plates, and four plate covers to support manual processing. However, you will have to purchase deep-well plates, standard plates, and tip combs in addition to that if you wish to process on a KingFisher ™ instrument.

Yes, you can, but we only recommend storing the lysed samples in Lysis Binding Mix at -20 degrees C for up to 4 days. Samples should be thawed to room temperature before proceeding with the RNA purification.

You can find scripts for using MagMAX™ mirVana™ Total RNA Isolation Kit with the MagMAX™ Express-96, KingFisher™ Flex, or KingFisher™ Duo instrument, on the product page.