mirVana microRNA Isolation Kit from Life Technologies

February 12, 2013

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University of Birmingham
School of Cancer Studies
Research Technician / PhD Student

An example of the reproducibility of multiple RNA samples after reverse transcription in a TaqMan QPCR assay. The RNA was isolated using the mirVana kit.

Company:

Ambion (Life Technologies)

Product Name:

mirVana microRNA Isolation Kit

Catalog Number:

AM1560

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In my work on viral and cellular gene expression I wanted to carry out qRT-PCR assays to quantify both microRNAs and primary transcripts on the same samples so I selected this kit to allow me to do so.

Experimental Design and Results Summary

Application

I use this kit to extract total RNA (including microRNA) from a range of cultured and primary cells. I then use these RNA samples for reverse transcription and TaqMan® quantitative PCR.

Starting Material

The samples I use include cultured lymphoma cell lines, epithelial cells and primary B cells. The samples have usually been previously infected with a DNA virus. My starting material can range from a few hundred cells to millions and the miRvana protocol is adaptable depending on the number of cells in the sample. I extract RNA immediately from freshly harvested cells (washed twice in PBS and pelleted) where the number of cells is low. Where the yield is likely to be higher, I wash and harvest the cells and then store at -80°C until I extract the RNA (usually within a week of harvesting).

Protocol Overview

This is a column-based kit where a homogenized cell lysate is first extracted using acid phenol chloroform and then the organic layer (after the addition of ethanol to optimize binding conditions) is applied to the column. The RNA species bind to the column fibers and surplus, non-bound cellular material is removed over three washes. The total RNA is then eluted in pre-warmed elution buffer or DEPC-treated water. There are additional steps that allow for the enrichment of small RNAs (>200bases) but I tend to use the total RNA as I am also interested in quantitatively measuring the messenger RNAs in my samples.

Tips

I have optimized my use of this kit quite a lot! This kit gives you a range of lysis buffer volumes (300-600 µl) to use depending on the number of cells in your sample. My pellets generally contain 0.3-3 million cells and over this range I find that 450 µl lysis buffer gives a good, consistent yield. Additionally, many of the downstream steps in the protocol are linked to this initial choice of lysis volume. If you use 450 µl, and therefore remove <480 µl after phenol chloroform extraction, you will find that you add 600 µl ethanol before you put the sample through the column. This is the perfect total volume to fit in a 1.5 ml microfuge tube (any larger and the sample has to be split, which is a hassle). When using the columns, I recommend that you spin them at low speeds, the column fibers may come free or the ‘fiber pad’ can distort. In a microcentrifuge, I carry out each spin at 8,000 RPM for only 3-6 seconds (which doesn’t actually reach the maximum speed). The final 1 minute spin and the elution (20 seconds) I also carry out at 8,000 RPM. You can buy this kit in two versions; one comes supplied with a small bottle of acid phenol chloroform, the other doesn’t. If you know a reputable and well-priced company to buy the phenol chloroform from, it is much cheaper to buy it separately! The elution buffer is pre-heated to 95°C but obviously the buffer will rapidly cool at room temperature. I apply the elution buffer directly from the heat block and then spin the columns immediately (place the heat block next to the microfuge). This will increase the yield of RNA. For low numbers of cells, you can reduce the elution volume by about half (from 100 µl to around 50 µl) to increase the concentration of RNA; however, this will decrease the overall yield. To maximize how much RNA you get, it is better to elute in 100 µl and adjust the amount of RNA you use in the reverse transcription reaction. Even when spinning samples at low speeds, fibers from the columns can become dislodged. There was a particular problem with this when the product was first launched and we had to request replacements for some of our badly affected kits. Although this issue has improved, there are still often a few fibers in the eluted RNA. These fibers interfere with spectrophotometer measurements of the RNA concentration and quality so if you notice this, spin the sample at full speed to pellet the fibers and then remove the RNA to a fresh tube, taking care to leave the fibers undisturbed.

Results Summary

The RNA from this kit is very good quality with consistently high yields. The kit also enables me to look at the expression of both messenger and microRNAs; however it can be more of a hassle than other kits I have used! I choose this kit over others when I have small numbers of primary cells as the yields are better than other kits I have used.

Additional Notes

This kit also does not have an on-column DNAse step unlike some others on the market so for assays which are not designed across a splice junction, an additional DNAse step is required after RNA isolation. The RNA isolated from this kit is not concentrated enough for use in Northern blotting – for this Trizol is better. Wash solution 1 always runs out first, the other solutions are supplied in huge excess, especially the lysis buffer, which means that it ends up going to waste.

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An example of the reproducibility of multiple RNA samples after reverse transcription in a TaqMan QPCR assay. The RNA was isolated using the mirVana kit.

Summary

The Good

Consistently high quality RNA with better yields than some kits and allows quantitation of all sizes of RNAs. The kit protocol can be carried out quite quickly as the spins are very short and can be optimized for small numbers of cells.

The Bad

Disintegrating columns!

The Bottom Line

Although this is still my kit of choice when dealing with small numbers of cells, for routine work, I would consider using a different kit.

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