MicroRNAs (miRNA) were first identified in the 1990s and ever since miRNA research has gained high significance. miRNA are short RNA molecules with an average length of 22 nucleotides. They perform a significant biological role in post translational modifications such as gene splicing. They act primarily as mRNA repressors and control translation. Traditional total RNA isolation can result in the loss of most of miRNAs due to their extremely small size. Isolation of total RNA including the smaller RNAs can be essential for several downstream applications such as microarray analysis and quantitative PCR analysis.
The miRNeasy Mini Kit for isolation of total RNA including miRNAs is a very convenient tool for isolation of good quality RNA in a relatively short time. The kit contains all the required reagents for the purification of total RNA. The most important ingredient in this kit which is not present in the regular RNeasy kit is the QIAzol lysis reagent. The lysis step using this reagent is essential for complete lysis of the cells and for inclusion of the smaller RNAs. Similar to the RNeasy Mini Kit, this kit can be used to isolate total RNA from a variety of specimens including animal cells and animal and plant tissues. The instruction book that comes with the kit is very informative.
We have used the miRNeasy Mini Kit in our laboratory for the last 6 months and have had excellent results. We were able to isolate miRNA from human cell lines such as neuroblastoma cell line SK-N-SH as well as from primary neural stem cells derived from fetal human brain. The most attractive feature of this kit is that it gives the user the flexibility to either isolate total RNA including the smaller microRNAs or obtain a relatively pure concentration of just the smaller RNAs. Using this kit, it is also possible to separate the larger RNAs (200 nucleotides) from the smaller RNAs. The procedure is very simple and is similar to the RNeasy protocol. However, the first step of lysing the cells requires the use of QIAzol lysis agent supplied in the kit. This reagent contains phenol, a highly toxic chemical and therefore care needs to be taken. This is followed by chloroform separation of the aqueous phase containing the RNAs. QIAzol lysis agent is very effective in not only complete lysis of the cells but also inhibits RNases and DNA contamination. This is particularly useful since DNA contamination can interfere with downstream applications such as gene quantification or microarray analysis. The rest of the protocol is column based, similar to the RNeasy kit.
Using this kit, we were able to isolate total RNA containing smaller miRNAs which we used both in microarray analysis as well as in qRT-PCR. Interestingly, we were able to get excellent results without enriching the smaller RNA fraction. A single procedure of isolating the total RNA was sufficient to get excellent microarray data.
In summary, the miRNeasy Mini Kit is a very easy and convenient kit for isolation of total RNA including miRNAs from a variety of cells and tissues.
miRNeasy Mini Kit from Qiagen
Excellent quality RNA can be isolated and separated into larger RNAs (>200 nucleotides) and smaller microRNAs.
The protocol involves use of QIAzol lysis reagent which contains toxic phenol.
The Bottom Line
This is an excellent kit for RNA isolation including smaller microRNAs.