mRNA Extraction

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 Invitrogen 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 Invitrogen 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 Invitrogen 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 Invitrogen 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.

• Learn more about how you can isolate mRNA in 15 minutes.

Bacteria lack the relatively stable poly(A) tails found on eukaryotic mRNA. Until very recently, isolating mRNA from bacteria has been virtually impossible. The Invitrogen 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.

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 Invitrogen™ 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.