A typical mammalian cell contains about 10–11 g of RNA, of which 1–5% is poly(A) RNA. The remaining RNA is mostly rRNA (80–85% of total) and low molecular weight RNA such as tRNA (15–20% of total). To separate the heterogeneous population of mRNA from the majority of the RNA found in the cell, Life Technologies offers two approaches for purifying mRNA from a wide variety of sample types. The first option starts with isolating total RNA by conventional means, followed by mRNA enrichment from the total RNA pool. The second option is to lyse cells and purify mRNA directly from the starting sample. Three distinct technologies used to accomplish this include: oligo(dT) cellulose/spin columns, oligo(dT)/magnetic beads, and coated plates. The information and links below will help you choose which methods and technologies are right for you.
Which mRNA Purification Kit is Right for You?
|Rapid mRNA purified from total RNA||No purification needed, mRNA direct from crude samples||mRNA from micro-sized samples
Best for next-generation sequencing of mRNA
|Flexible system, isolate mRNA directly or enrich from total RNA||HTP mRNA directly or enrich from total RNA|
|Prep time||15 minutes||15 minutes||15 minutes||1 hour||1.5 hours|
|Direct Isolation of mRNA from crude samples||No||Yes||Yes||Yes||Yes|
|mRNA Enrichment from total RNA||Yes||No||No||Yes||Yes|
|Amount of starting material||< 75 ug Total RNA||
< 20x106 cells
2 - 200 mg animal tissue
4 - 400 mg plant tissue
|< 1x104 cells
< 5 mg plant or animal tissue
|< 1x108 cells
50 mg plant or animal tissue
2 - 400 µg Total RNA
|100 to 1x106 cells
< 4 mg tissue
40 µL whole blood
100 ng - 100 µg Total RNA
|Format||Fast magnetic bead capture from pure total RNA||Fast magnetic bead capture directly from crude samples||Fast magnetic bead capture directly from micro-size samples||Convenient affinity bead spin column
(no magnet required)
|HTP ready 96-well coated plate|
|High throughput compatible||No||No||No||No||Yes|
|Kit Size||2mL oligo dT beads||5mL or 10mL oligo dT beads||2mL oligo dT beads
(sufficient for 100 mRNA preps)
|20 preps||96 or 960 preps|
|Kit Price||$467||$484 or $730||$587||$528||$276 or $2,161|
|Order Now||Order Now||Order Now||Order Now||Order Now|
Column or Magnetic Bead mRNA Extraction and Enrichment Kits
Oligo(dT) Cellulose/Spin Column Methods
The rRNA contamination that plagues mRNA enrichment is not exclusively caused by nonspecific adsorption to the oligo(dT) matrix, but also by binding to, and co-purifying with, messenger RNA (mRNA). Premeasured oligo(dT) cellulose combined with novel reagents and protocol improvements in a familiar and readily adoptable spin column format help minimize these unwanted interactions while still promoting efficient oligo(dT) selection.
This methodology has been incorporated into the Ambion® Poly(A)Purist™ and MicroPoly(A)Purist™ Kits. The result is an mRNA selection procedure that can be completed in approximately 2 hours and typically requires only a single round of selection, yet yields RNA that is more highly enriched and devoid of rRNA. The Poly(A)Purist™ Kit is optimized to select poly(A) RNA from total RNA in a very low–ionic strength buffer (it cannot be used with crude cell or tissue lysates), whereas the MicroPoly(A)Purist™ Kit can use total RNA (as well as small amounts of tissue or mammalian cells) as starting material and includes its own lysis step.
The Life Technologies FastTrack® 2.0 mRNA Isolation Kit allows isolation of mRNA directly from cells and tissue or enriches mRNA from purified total RNA. The Micro-FastTrack™ 2.0 mRNA Isolation Kit purifies mRNA directly from smaller inputs of cells and tissues or enriches mRNA from smaller inputs of purified total RNA. Both protocols are completed in 2 to 3 hours without the need for ultracentrifugation or guanidinium lysis.
Oligo(dT)/Magnetic Capture Methods
Column methods for mRNA purification are reliable but require extensive manual interaction, which means more time and reduced throughput. Magnetic beads conjugated with oligo(dT) offer additional handling and scaling benefits; however, this procedure does require the use of magnetic stands.
The Ambion® Poly(A)Purist™ MAG Kit employs the same hybridization and wash solutions as those in the Poly(A)Purist™ Kit but with the added ease, speed, and scalability of magnetic beads. The protocol typically takes only 45 minutes, and the kit performs from 8 large enrichment reactions (up to 1 mg total RNA input) to 80 small enrichment reactions (as little as 30 µg total RNA input). Like the Poly(A)Purist™ Kit, this kit only enriches mRNA from previously purified total RNA.
The Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA Preps, enriches mRNA from purified total RNA, typically in 15 minutes, and allows users the flexibility to elute the mRNA from the beads in as little as 5 µL, or even to perform cDNA synthesis directly on the beads without elution.
For those who prefer using Dynabeads® technology with sample lysis and enrichment in one kit, the Dynabeads® mRNA DIRECT™ Kits enable 15-minute lysis and enrichment processing of a variety of sample types. In fact, it is possible to capture mRNA and generate cDNA libraries from a single cell using this technology. The flexible format allows users to scale up or down and gives the option to elute the mRNA from the beads in as little as 5 µL, or to perform cDNA synthesis directly on the beads without elution. The 5 mL size is sufficient for 20 standard isolations, while the 10 mL size is sufficient for 40 standard isolations.
For those who work with smaller inputs, the Dynabeads® mRNA DIRECT™ Micro Kit provides enough reagents for 100 mRNA isolations from up to 2.5 x 104 mononuclear cells, up to 1 x 104 cultured cells, or up to 5 mg tissue (depending on the tissue), with the same elution flexibility as the other Dynabeads® kits.
The Life Technologies FastTrack® MAG family comprises three kits: a 12-reaction micro kit, a 6-reaction maxi kit, and a 2 x 96-well plate kit. All versions of the FastTrack® MAG kits support both lysis and enrichment or enrichment only from cells, tissue, or total RNA via an oligo(dT)–magnetic bead method. The FastTrack® MAG Micro mRNA Isolation Kit is optimized for smaller inputs (<50 µg total RNA, <106 cells, and <50 mg tissue), while the FastTrack® MAG Maxi mRNA Isolation Kit handles larger inputs (<2,000 µg total RNA, <5 x 107 cells, and <500 mg tissue). The FastTrack® MAG 96 mRNA Isolation Kit utilizes a semiskirted 96-well plate prefilled with oligo(dT) magnetic beads and processes up to 20 µg of purified total RNA or 1 x 105 cells. All of the FastTrack® MAG Kit protocols can typically be performed in 1–1.5 hours.
Bacteria lack the relatively stable poly(A) tails found on eukaryotic mRNA. Until very recently, isolating mRNA from bacteria has been virtually impossible. The MICROBExpress™ Bacterial mRNA Isolation Kit employs a novel technology to remove up to >95% of the 16S and 23S rRNA from total RNA of E. coli and other bacterial species. The kit is suitable for mRNA enrichment from ≤10 µg of purified bacterial total RNA in about 2 hours from a broad spectrum of gram-positive and gram-negative bacteria. mRNA isolated with the MICROBExpress™ Kit is a superior template for synthesizing labeled cDNA for array analysis and is ideal for quantitative RT-PCR, northern blotting, and cDNA library construction.
mRNA Extraction and Enrichment via Coated Plates
The most distinctive mRNA purification method takes advantage of specially treated plates in conjunction with optimized reagents to both lyse and enrich mRNA from cells, tissue, blood, or purified total RNA. The mRNA Catcher™ PLUS product utilizes a proprietary surface treatment of a 96-well plate to facilitate easy and automation-friendly mRNA purification. Locked Nucleic Acid (LNA) technology incorporated into the 20-mer oligo(dT) increases specificity and hybridization efficiency while removing centrifugation, precipitation, and phase-separation steps from the overall workflow.
The mRNA Catcher™ PLUS Kit is capable of handling 100 ng to 100 µg total RNA, 100 to 106 mammalian cells, up to 4 mg tissue, and 40 µL whole blood. The binding capacity is >80 ng/well. The protocol employs a simple lysis, hybridization, wash, and elution process that can be completely automated on open-format liquid handlers in approximately 1.5 hours from start to finish. The system also has the flexibility to elute mRNA or to perform cDNA synthesis directly in the hybridization plate without elution.