RNeasy Mini Kit From Qiagen

The RNeasy Mini Kit by Qiagen is used to purify RNA from animal cells or tissues, yeast and bacteria. It comes with RNeasy mini spin columns, collection tubes and RNase-free reagents and buffers. It also comes complete with a protocol booklet that outlines the number of cells than can be used from each tissue or cell type, and different protocols depending on what machine (microfuge or vacuum manifold) is being used for the extraction method.

Part of my work consists of infecting human cells with bacteria and then extracting either the human or bacterial RNA (both types of RNA can not be extracted from the same sample) from a particular infected sample. I primarily use the RNeasy Mini Kit and a benchtop centrifuge to do this. When extracting human RNA, I use the RNeasy Mini Kit directly, but when extracting bacterial RNA, I first have to extract RNA using a phenol chloroform based method before using the RNeasy Mini Kit so as to remove any trace of human RNA from that sample as this kit will not differentiate between types of RNA. However, both methods yield high quality RNA which is not degraded, even though the bacterial RNA has already been subjected to a phenol chloroform extraction. I am not always careful about the number of cells I use to begin with, but have never had any problems in extraction; however, if the cell pellet is very large, then I double the volume of RLT lysis buffer and run the sample through the column twice, or spilt the sample in two. Otherwise, the filter in the column becomes blocked and material can be lost.

The protocol is very simple and easy to follow. It begins with a lysing reaction to break open the cell or tissue material. This is then followed by a few washing steps using buffers provided in the kit. Each step requires the samples to be spun down in a bench top microcentrifuge for only 15 – 30 seconds. I also always include the optional steps mentioned in the protocol, like the removal of small amounts of DNA using the RNase-Free DNase Set (cat #79254, also from Qiagen). This step adds 20 minutes to the protocol, but removes any trace of excess DNA very effectively.

One useful tip I would give to any future user would be to perform the elution step twice as this has been shown to increase the RNA yield significantly. After the first elution step, I remove the collected sample and reapply it to the filter once more. Also, while the manufacturer says that an elution volume of between 30 – 100 ìl can be used, I have never used more than 40 ìl of water and have had problems in downstream applications when using any other elution buffer.

It is very important to eliminate any trace of the ethanol present in the wash buffers from the sample so I always include the optional steps in the protocol that remove the wash buffers. However, there are not enough extra 2 ml collection tubes provided for this. I use our own 2 ml tubes, not 1.5 ml tubes, because the 2 ml tubes allow the wash buffer to completely clear the column thus, the wash buffers are more effectively removed.

I have found this product very efficient in the purification of RNA and it has always given me high yield of good quality RNA. The kit is easy to use and doesn’t require many additional components, making it a very convenient extraction method.