Alternative Protocols for the Isolation of RNA
The Covid-19 pandemic has spurred mass diagnostic testing of populations around the world. The explosion in RNA testing, related to the pandemic, has caused significant shortages of RNA isolation reagents as stocks are currently allocated for testing needs. The shortage may very well continue as governments around the world plan to re-open shuttered economies, which will be dependent upon not only continued diagnostic testing but also surveillance.
For researchers not directly involved with the surveillance of Covid-19, who find themselves without RNA kits, OPS Diagnostics is introducing an alternative. The Synergy™ nucleic extraction chemistry, originally developed for plant DNA isolation, has successfully been used for total nucleic acid extraction and RNA isolation.
The Synergy™ nucleic acid extraction chemistry depends on a bead beating homogenization step. These kits are designed to be used in all bead beaters from simple bead beaters to high throughput machines that hold deep well plates. RNA isolated using bead beating has shown to be of high quality when analyzed on the Bioanalyzer, and suitable for most downstream applications.
The development team at OPS Diagnostics has been working on adapting Synergy™ for RNA isolation. The existing Synergy™ products were designed for DNA isolation, thus modification using several important, but obtainable, components is required.
Most importantly is the addition of dithiothreitol (DTT) to the homogenization tubes. The team has found, that by the addition of a reducing agent (e.g., mercaptoethanol or dithiothreitol) sufficiently inactivated RNases during isolation. Then following the first elution from the spin column, result in total nucleic acid isolation; the eluents need to be treated with DNase and a second spin column purification procedure, to isolate purified RNA. Some applications, used these modifications, to isolate of RNA from cell culture, quantify yeast transcripts, and detect RNA viruses in plants.
This protocol uses 2 ml Synergy™ homogenization tubes supplemented with dithiothreitol. The sample size ranges from 10 to 50 mg, or 100 µl or less for aqueous samples. Additional equipment required a bead beating homogenizer, such as the HT Mini™ and a microcentrifuge. The protocol first isolates total nucleic acid, the option is there to stop at total nucleic acid isolation and running the protocol to completion removes the DNA to yield purified RNA.
Modified Synergy™ RNA/Total Nucleic Acid Isolation Protocol
- Add up to 50 mg solid or 100 µl aqueous sample, 500 µl of Plant Homogenization Buffer, and 10 µl of 1 M DTT to the 2 ml homogenization tube.
- Place the homogenization tube into a bead beater and homogenize the sample at the highest speed for 1 minute. If the sample is not completely homogenized, repeat the process, as the sample can occasionally press against the tube wall and avoid homogenization. When adequately processed, the tube will lack foam.
- Centrifuge the homogenization tube at 15,000 x g for 5 minutes to pellet the debris, grinding resin and contaminants.
- Transfer the clear supernatant into a clean centrifuge tube.
- Add 7/10 volume of 100% isopropanol. Vortex. Incubate at -20°C for 15 minutes.
- Transfer the solution to a spin column placed in a collection tube. Centrifuge the column at 8,000 x g for 1 minute to bind the DNA to the column.
- Wash the column with 250 µl of ice cold 70% ethanol. Centrifuge the column at 8,000 x g for 1 minute to pass through the wash solution. Repeat the wash.
- Elute the total nucleic acids by placing the column in a clean collection tube. Add 50 µl of Molecular Biology Grade Water and centrifuge the column at 15,000 x g for 1 minute. This can be stored at -80°C
- Add 5.5 µl of DNase 10x Buffer and 2 µl of DNase to samples. Incubate at room temperature for 10 minutes.
- Add 10 µl of 1 M Tris, pH 8 and 2.5 µl of 1 M NaCl.
- Repeat steps 5-8. Final elution can be made in either Molecular Biology Grade Water or TE Buffer. Store the samples at -80°C
The high throughput Synergy™ kit can also be adapted for total nucleic acids and RNA isolation. This protocol requires a plate homogenizer, such as the Geno/Grinder® or HT Homogenizer II™. The wells, of 96 well plate, are smaller, thus less sample can be homogenized as compared to 2 ml disruption tubes. The protocol first isolates total nucleic acid, the option is there to stop at total nucleic acid isolation and running the protocol to completion removes the DNA to yield purified RNA
RNA Isolation Protocol
- Remove the strip caps and add up to 40 mg of sample to each well of the 96 Well Synergy™ Homogenization Plate, followed by the addition of 350 µl of Plant Homogenization Buffer and 7 µl of 1 M DTT. (If the entire plate is not being used, evenly distribute the strips across the plate. At least two strips at opposite ends of the plate are necessary.)
- Reseal the wells with the strip caps. Press the seals in firmly so they are even across the plate.
- Place the Well Support Mat underneath the homogenization plate to support wells during grinding. Homogenize using a plate homogenizer (e.g., GenoGrinder® or HT Homogenizer II™) for 10-15 minutes at 1,500 rpm.
- Centrifuge the plate for 10 minutes at 2,100 x g.
- Transfer up to 180 µl of supernatant from each well to the Filter Plate. Place a Collection Plate under the Filter Plate and centrifuge for 10 minutes at 2,100 x g.
- Add 120 µl of isopropanol to the solutions, mix and incubate at -20°C for 15 minutes.
- Place a new Collection Plate under the Binding Plate. Transfer the lysates to the Binding Plate and centrifuge for 10 minutes at 2,100 x g. Discard the filtrate from the Collection Plate and move the plate back under the Binding Plate.
- Wash the bound nucleic acid by adding 200 µl of ice cold 70% ethanol to each well. Centrifuge for 5 minutes at 2,100 x g. Discard the filtrate from the Collection Plate and place the plate back under the Binding Plate.
- Repeat the wash (step 9). Centrifuge for 5 minutes at 2,100x g.
- Replace the Collection Plate with an Elution Plate.
- Elute the nucleic acid by adding 50 µl of Molecular Biology Grade Water or TE Buffer. Centrifuge for 10 minutes at 2,100 x g.
- Add 5.5 µl of DNase I 10x Buffer and 2 µl of DNase to each sample and mix. Cover the wells and incubate at 37°C for 10 minutes.
- Add 10 µl of 1 M Tris, pH 8 and 2.5 µl 1 M NaCl to each sample and mix.
- Add 43 µl of isopropanol to each sample, mix and incubate at -20°C for 15 minutes.
- Repeat steps 8 through 12. Final elution can be made in either Molecular Biology Grade Water or TE Buffer.
- Cover the Elution Plate with the provided lid, seal with parafilm and store at -80°C.