Tips and tricks for isolation of RNA from cells and tissues remains a very popular subject. I can’t tell you how many discoveries in science depend on identifying that elusive low-copy mRNA with the 5 second half-life!
Joking aside, we’ve covered quite a few of the basics so far. For example, I’ve explained how a strong and complete homogenization in denaturing lysis buffers performed very quickly is the key to denaturing nucleases and releasing the maximal amount of RNA. And we’ve talked about the options for homogenization of samples, beginning with liquid nitrogen and ending with all the types of beads available for bead beating using high powered instruments. All the methods work, so the choice comes down to what is available to you and how many samples you have to do in a day. Bead Beating can easily process many samples at one time with equal and efficient lysis, so consistency between preps is maintained. Methods that are performed one sample at a time, such as mortar and pestle or rotor-stator homogenizers are better if only a few samples are involved but increase the risk of cross-contamination if cleaning the equipment between the samples is required.
One topic we haven’t discussed yet is how to analyze your RNA quality and quantity. Accurate analysis of your RNA is critical for ensuring repeatable results in the next steps. Consistency begins at the very start.
Measuring RNA Yields:
There are several methods for quantifying your RNA. The most frequently used method and the least expensive is using a Nanodrop or similar type UV absorbance measuring instrument. The Nanodrop has the advantage of providing you information on RNA purity. You’ll get your 260/280 ratios, which measures the level of protein contamination in the sample, and the 260/230 ratio which tells you whether contaminants from the prep are in the sample (guanidine salts). For RNA, the ideal 260/280 ratio is between 1.8-2.1 and the 260/230 ratio ideally is above 1.5. Below is an example of the type of information provided by the Nanodrop.
Keep in mind that if RNA yields fall below a certain level (20 ng/ul), the Nanodrop readings are not quite accurate. We find that the RNA peak at 260 is not significantly high enough to balance the 280 or 230 readings, resulting in ratios that may not look right. In this case, take a look at the wavelength plot data on the Nanodrop. Make sure you are not seeing a peak at 220-230 or at 280. If the only bump in the curve is at 260, your RNA is ok.
To give you an example of what I mean, here are some wavelength plots from a soil RNA experiment. In this experiment you see the two curves that are peaking high at 230 and then coming down like a ski slope. This is not good. The high peak at 230 usually indicates salt contamination which typically comes from guanidine thiocyanate or other similar compounds used in RNA preps to lyse cells and inhibit RNases. If not washed out completely, these will affect the entire spectrum through the wavelengths and lead to false high 260 readings.
The yellow and green curves are example of samples with a lot of both salt and humic acid inhibition. Humic acids will also absorb at 230 and continue throughout the spectrum of wavelengths, peaking at 320. All of these first four samples were brown at the end of the isolation.
The correct looking plots are the ones shown below the green and yellow curves. Here we see a nice downslope at the 230 reading followed by the peak at 260 and then the curve goes down again at 280. If we look at the very bottom two samples, in red and black, you can see what would happen if you had very pure RNA but a low concentration. Now the 230 and 280 readings are low such that there is no longer a 2:1 ratio between the 260/230 or 260/280. This doesn’t mean the RNA is not good quality. As long as we don’t see a ski slope or the curve going straight across, this RNA is ok to use.
However there are other ways to measure RNA yields with greater sensitivity at low concentrations. We sometimes use the Ribogreen kit with our Qubit instrument. The advantage of Ribogreen dye for quantification is that it measures the RNA content only. While this is very convenient, it does not give you any information about RNA integrity or purity. We’ve covered the difference between UV absorbance vs fluorescent dye in quantifying DNA in a previous article using plasmid preps as an example. For more comparison data on this, check out the article linked above.
Besides knowing your yields of RNA, the other key factor for determining if your RNA prep worked well is looking at integrity. Integrity means, how intact and undegraded is the RNA. Traditionally, we determine this by looking at the intensity of the rRNA bands on an agarose gel. In eukaryotic cells, the 28S should be double the intensity of the 18S band an in bacterial cells, we look at the 23S in relation to the16S rRNA bands. To check RNA integrity, we simply run 5-10 ul of the sample on a standard 1% DNA agarose gel in 1X TAE buffer. This works very well for checking RNA. A denaturing gel is not necessary for doing a quick check. Here is an example of how the RNA gel should look with RNA from mouse liver using our PowerLyzer UltraClean Tissue and Cells RNA Kit.
In this example, 5 ul of RNA was run because liver has very high yields of RNA. If you overload the gel, you will not get good clear separation of the bands.
With high quality RNA, the upper band (28S) should look double the intensity of the lower band (18S) and appear crisp and sharp. If the homogenization was performed well, DNA in the upper part of the gel should be absent. If the upper band 28S appears to be equal intensity to the lower band, it indicates some level of degradation has occurred. Smearing between the 28S and 18S is normal as this is where most of the mRNA will migrate. Smearing far below the 28S band is never a good sign.
There is another way to analyze RNA integrity and that is using the Agilent Bioanalyzer or similar type instrument. This instrument is nice because it uses only 1-2 ul of RNA and it measures the sizes of the rRNA bands to deliver what is called a RIN number. The RNA Integrity Number (RIN) is a way to standardize the quality of the RNA between preps.
Here is an example of an Agilent Bioanalyzer result. These are liver RNA samples analyzed with the Eukaryotic Total RNA Nano Series II kit. The RIN numbers are shown for two samples processed using bead beating and then purified using the MO BIO UltraClean Tissue and Cells RNA Kit or a Competitor’s kit that also uses a spin filter.
The sizes coming from the Bioanalyzer are smallest to largest, left to right. The peak between 45-50 seconds is the 28S rRNA and the second peak around 41-42 seconds is the 18S. If the RNA were degraded, we would see some blips or peaks on the scan around 35-40 seconds and earlier. The larger the RNA, the later it appears. If genomic DNA were contaminating the RNA, it would appear as a peak on the right.
Greater detail on both the Nanodrop and Agilent Bioanalyzer can be found in this helpful document put together by Biomedical Genomics.
Not everyone has the ability to run the Agilent Bioanalzyer in their lab. The disadvantages are that it is a very expensive instrument and the kits for analyzing RNA are also very expensive. If you have a core facility on campus, they may allow you to run samples on it, but they’ll usually charge a price to offset the cost of the kits.
But that’s why you have options. Using an agarose gel to check integrity and presence or absence of DNA and then the nanodrop for checking purity and yields, you’ll be able to discern whether you have high quality and yields of RNA.
And one last note about DNA contamination….
DNA contamination in the RNA can be a hassle, especially for those of you working in microbial genetics, where primers cannot be designed to cross intron-exon boundaries. On-Spin Column DNase removal systems are one effective way to remove the DNA before elution of RNA. By letting the DNase soak into the membrane, it works to digest and remove the DNA before the RNA is eluted. But often times it is not enough. If the sample or cells were stored in RNALater or RNAProtect Bacteria Reagent, the DNA will become much more resistant to DNase treatment. In these cases, DNase treatment in solution is the better way to go, where the DNase has full access to the DNA instead of trying to work around the confines of a silica membrane. The disadvantage of using DNase in solution is that it must be inactivated before PCR using either EDTA and heat. To overcome this problem, we developed the RTS DNase Kit, a room temperature stable high velocity DNase enzyme that is easily removed using a DNase binding resin. It is extremely gentle on your RNA and highly effective at removing the DNase.
Samples are available:
Did I mention that we have samples of almost every kit we make? If you want to sample any of our RNA kits or the RTS DNase, free samples can be requested online or through contacting technical support.
MO BIO now offers RNA kits optimized for 10 different samples types. Your sample is unique so why shouldn’t your kit be too? Some of these kits have our IRT method for removing inhibitors and others do not, but they are all optimized, saving you time from troubleshooting, and saving you time for life!
Recent publication: UltraClean Tissue & Cells RNA Isolation Kit
Splice-Mediated Motif Switching Regulates Disabled-1 Phosphorylation and SH2 Domain Interactions
Zhihua Gao, Ho Yin Poon, Lei Li, Xiaodong Li, Elena Palmesino, Darryl D. Glubrecht, Karen Colwill, Indrani Dutta, Artur Kania, Tony Pawson, and Roseline Godbout
Mol. Cell. Biol., Jul 2012; 32: 2794 – 2808.