RNA isolation. It doesn’t get that much hype, but it should considering it’s the first, and perhaps most critical, step in virtually every RNA analysis workflow out there. Today’s favorite techniques are increasingly accurate and precise and some applications, like next-gen sequencing, will have you swimming in data analysis for weeks. That’s not such a bad thing as long as it’s good data.
We hate to break out the old “garbage in; garbage out” adage, but it really does apply here. And that’s especially true for researchers focused in small or non-coding RNAs, like miRNAs, that only represent a small fraction of sample’s total RNA.
The reigning RNA quality champ is the TRI-Reagent® approach (or other acid- guanidinium-phenol variation). But, while the reagent itself works well, there’s plenty of room for improvements.
RNA Separation Anxiety
Mixing your cells, tissue or biological fluid with the isolation reagent is the easy part. The rest of the steps are where the problems begin to creep in.
- Speed: The first problem is that the phase separation/precipitation technique is slow. It can take over an hour to complete, and the cumbersome manipulations can put the brakes on as well if there are multiple samples to handle.
- Phase separation: It takes a steady pipette hand to get every last microliter of the RNA phase from the tube. If you leave some behind, you’re wasting precious sample; if you take too much, there may be some phenol carryover. Not only that but you can’t tell how well you’re capturing miRNAs. None of these situations are good.
- Precipitation & re-suspension: During these steps, at some point you’ll probably be asking yourself: Hey, where’s the pellet? Not exactly confidence inspiring, and the worst part is that you won’t really know until later when the data comes in.
Phasing Out Unwanted RNA Prep Steps
Recently some enterprising members of the Zymo Research R&D team introduced a new spin (column, that is) to replace the most undesirable aspects of phase separation and precipitation protocols. So now you can just add your TRI-Reagent®/sample mixture to a column, spin, wash and elute with their new kit, Direct-zol™ RNA MiniPrep. Crafty.
Whether it’s sliced bread, or wheels for suitcases, the best ideas will click immediately when you see them. But just in case you don’t quite buy it yet, just check out what switching over to the specially designed Zymo spin columns can do for your isolations.
- Speed: Preps now take only 10 minutes instead of an hour or more, with a whole lot less handling. “It cuts down on time considerably and enhances the output and quality of RNA isolated by less skilled student researchers.” Says A. Underwood from Walsh University.
- Phase separation and Precipitation/re-suspension: Gone.
- Sample or small RNA species loss: Not anymore. Comparisons show better total yields and increased smallRNA and miRNA recovery (about twice the <200 nucleotide fraction, and 4 times the RNA with <40 nucleotides). According to Kimberly N. from Duquesne University “I found that the kit gave about 30% more RNA and the sample was cleaner when run on a gel.”
- Better RNA quality: Using spin column purification gives you a broad size-range of high quality, DNA-free RNA without phenol of crossover contamination. “When I took OD readings of my samples I was very pleased with the 260:280 ratio and the quality of the RNA.” Commented Emily D. from the University at Buffalo She’s not alone. “The ratio 260/280=1.98, 260/230=1.89, RIN=9. Excellent for expression arrays!” exclaims J. Wen at The Methodist Hospital Research Institute.
J. Hicks at North Carolina State University sums it up pretty well. “We purify RNA almost every day in our lab, we look at both mRNA and microRNA expression using a variety of downstream applications. Overall, I liked the kit; it is much faster than the traditional TRIzol® protocol…” he went on to say. “I like the fact that I can use samples stored in TRIzol®, as I often do this when I have large time course cell culture studies.”
So, how can you get this RNA purification breakthrough into your lab? The good news is that you can get all of these new enhancements in once box. Just head over the Zymo Research website to check their new Direct-zol™ RNA Miniprep Kits.