MEGAclear™ Kit From Applied Biosystems

A quick clean-up of any large scale transcription reaction can be very laborious, time consuming and a multi-step protocol is often needed. In addition, great flexibility in regards to downstream applications is not easy to achieve, but often considered necessary. One way to clean up RNA is to use different precipitation methods combined with various washing steps.

The MEGAclear™ Kit from Applied Biosystems includes a binding solution concentrate, wash solution concentrate, elution solution, filter cartridges, and collection tubes, with or without user manual, which can also be downloaded. For the preparation of the wash buffer, the user needs to add 100% ethanol (ACS grade or better) to the wash solution concentrate before use. In addition, 100% ethanol is added to the sample before pipetting it onto the filter for ensuring RNA binding to the filter cartridge. A microcentrifuge capable of attaining 10,000-15,000 x g or a vacuum manifold is also required.

In my laboratory routine, I have used this kit for different applications. An efficient separation of RNA was achieved after in vitro transcription reactions, RNA amplification reactions (aRNA), capping reactions, and from post-labeling reactions. Subsequently, the RNA was used for different downstream applications requiring high purity RNA.

One of the major advantages of this kit is the fast and easy removal of unincorporated nucleotides, short oligonucleotides, proteins, and salts from RNA. The spin column approach ensures easy handling and a quick protocol, usually less than 30 minutes.

The protocol is quick and easy to follow. The sample obtained from various upstream reactions is brought up to 100 ul by adding elution solution. After adding binding buffer and 100% ethanol, the sample is loaded into one of the supplied filter cartridges to bind the RNA. The RNA mixture is passed through the filter using either a microcentrifuge or vacuum device. After two subsequent washes, the RNA is eluted by using the included elution buffer, which will release the RNA from the filter. The protocol gives two options for the elution step, depending on the laboratory equipment. The RNA can be eluted by using heated elution buffer (95ºC) or by applying the elution buffer to the filter and placing the filter in a heat block (65-70ºC for 5-10 minutes). In my hands, either of the two possible approaches worked in an equal way and with the same efficacy. To maximize the RNA recovery, the elution procedure can be repeated once, eluting the RNA into the same tube again. The kit recovers RNA from 1 ng to 500 µg. One of the kit’s limitations is the size of RNA that can be recovered. Purifications of ssRNA larger than 100 nt and dsRNA larger than 200 bp is possible. Therefore, the kit is not suitable for the purification of smaller constructs. However, in our lab a 75 nt ssRNA piece was successfully purified using the MEGAclear™ kit.

Overall, I think the kit is working very well, is quick and easy to use.