Quantcast

From Trizol to RNA in a Heartbeat: Zymo Research’s Direct-zolTM RNA MiniPrep Kit Makes RNA Isolation a Breeze

Ah, the woes of RNA isolation. Are you tired of trying to conquer the perfectionist phenol-separation? Do multiple precipitation steps cause you nightmares? Do you wake up in the morning in a cold sweat, dreading the approaching RNA purification day? Seriously, who needs to go to the gym when you can stay fit just by attempting to wrench those hundreds of RNA samples from the sticky proteins and persistent DNA? Ok, let’s admit it, RNA samples can be very precious and you want to get clean, unbiased, and concentrated RNA samples quickly and reliably!

The TRIzol method for RNA extraction has been the gold standard. TRIzol is a pink colored chemical solution, which was first applied for RNA isolation by Piotr Chomczynski and Nicoletta Sacchi in 1987. TRIzol acts as a powerful protein denaturant to break down cellular components and inactivate all enzymes, including RNases. TRIzol extraction typically uses acidic phenol-chloroform to confine total RNA in a clear aqueous phase, while proteins and cellular debris end up in the pink organic layer. RNA can be recovered by precipitation with ethanol, washed, and then dissolved in water. You can find more detail about this method of RNA isolation here.

However, TRIzol RNA extraction method has several serious limitations. It can get rather tedious due to many steps, making it hard to use in high-throughput manner. The process requires a phase separation step by phenol-chloroform extraction as well as a number of precipitation steps. This takes a long time to perform (anywhere from one-half to several days). The phase separation step can be tricky and, if done incorrectly, can lead to protein contaminated RNA sample. Moreover, phenol carryover can be an issue and interfere with reverse transcriptase steps down the line. Even worse, it could lead to a biased RNA sample, because phase separation selectively enriches for some species of miRNA (1). Therefore, the traditional TRIzol approach can be daunting to new users. Luckily, Zymo Research’s Direct-zolTM RNA Miniprep Kit can help you overcome all these problems. It combines the power of organic extraction reagent with speed and simplicity of spin-column purification.

Are we there yet? This is taking forever!

The entire process takes less than 10 minutes to complete! In fact, you can get purified RNA directly from your TRIzol sample in 7 minutes. Compare this to the more traditional methods that would take hours. All you have to do is simply apply your sample in Trizol to the Zymo-Spin column, then spin, wash, and elute. Can you believe it’s that easy?

Figure 1Figure 1: High-quality small and large RNAs are effectively recovered using the Direct-zol™ kit compared to kits from other suppliers.

 

Bada Bing Bada Boom: It’s pure and simple.

Because there is no phase separation, there is no phenol carry over and no contamination. If you are concerned about that pesky DNA in your RNA samples, you have the option of performing on-column DNase treatment. Moreover, there are no precipitation steps to worry about. You can process multiple samples in parallel, with one sample per column and all washes performed on the same column.

Figure 2
Figure 2: Direct-zol™ RNA isolation protocol

The system also allows for easy high-throughput and fully automated RNA isolations. The eluted RNA is suitable for a variety of downstream applications. So, let’s take a look at the protocol:

Step 1. Add ethanol to tissue/cells sample in TRIzol, TRI Reagent, etc. You can basically start with any TRIzol stabilized sample, including cells, tissues, in vitro reactions, tough-to-lyse samples, FFPE, plants, microorganisms, and body fluids.

Step 2. Mix and add the entire sample to the unique Zymo-Spin™ column to bind RNA. No phase separation or precipitation needed. You can pinch yourself, this is for real! This gives you the ability to purify up to 100 µg (per prep) of high-quality RNA per column.

Step 3. Wash spins are carried out in the same column at room temperature, so no need to fight your lab mates over that coveted refrigerated centrifuge.

Step 4. Finally, simply elute your DNA-free, phenol-free, and hassle-free total RNA in RNase free water!

 

Unbiased: Just like how science should be

Those of you with your mouths gaping, snap out of it—it’s really true! But let’s ask some important questions: Is there bias towards miRNA? Nope, this little gem of a kit is unbiased towards miRNA unlike other traditional methods (1). The columns are engineered to purify all RNAs 17 nucleotides in length or longer. Even when compared to other kits that are designed to enrich for small RNAs, Direct-zolTM RNA kit allows for isolation of smaller RNAs that are difficult to isolate. All of this without the danger of any carry-over due to phase separation! Just your clean, DNA-free, protein-free RNA. Here is your chance to jump on your lab bench and yell “Freeeeeeedom!”

Figure 3.
Figure 3. The data show RNA purified from TRIzol® samples using the Direct-zol™ RNA MiniPrep compared to an unbiased method (mirVana™, Ambion). Micro-RNA analysis was performed using miRNA-Seq (MiSeq®, Illumina) and a direct hybridization assay (nCounter®, Nanostring).

 

Finally, because of the fast processing time and scalability, Direct-zolTM RNA MiniPrep kit can be used to isolate RNA for further downstream analysis with RT-PCR, transcription profiling, hybridization, sequencing and other applications. It seems particularly useful for researchers focusing on the role of small and large RNAs in the regulation of gene expression by message silencing and the role of RNAs in epigenetics.

Learn more by watching the below video:

 

 

The Direct-zol™ RNA Purification system comes in a Microprep, Miniprep, and Miniprep Plus format along with high-throughput and automated options.

What are you waiting for? More information on Zymo Research’s Direct-zolTM RNA MiniPrep kit can be found here.

References

1. Kim et al (2012) Molecular Cell 46(6):893-895