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Obtaining high-quality RNA is the first, and often the most critical, step in performing many molecular techniques such as reverse transcription real-time PCR (RT-qPCR), transcriptome analysis using next-generation sequencing, array analysis, digital PCR, northern analysis, and cDNA library construction. To generate the most sensitive and biologically relevant results, the RNA isolation procedure must include some important steps before, during, and after the actual RNA purification. The following application note discusses various best practices in sample collection, storage, and RNA isolation procedures for maximizing the yield and quality of sample RNA, many of which involve the use of Ambion® technologies.

Optimizing RNA Preparation and Analysis

Our ongoing research into optimizing RNA preparation and analysis has identified several points in the process that can commonly be improved and are often overlooked:

  • Treatment and handling of samples prior to RNA isolation
  • Choice of technologies used to prepare the RNA
  • Storage of the prepared RNA sample

Most traditional RNA isolation procedures take place in the presence of RNase inhibitory agents (typically strong denaturants like guanidine salts, sodium dodecylsulfate (SDS), or phenol-based compounds that are designed to lower the risk of RNA degradation in a sample). However, it is typically prior to and after the isolation when RNA integrity is at highest risk.

Step 1: Sample Collection and Protection

Finding the most appropriate method of cell or tissue disruption for your specific starting material is important for maximizing the yield and quality of your RNA preparation. During sample disruption for RNA isolation, it is crucial that the lytic agent or denaturant be in contact with the cellular contents at the moment that the cells are disrupted. This can be problematic when tissues or cells are hard (e.g., bone, roots), when they contain capsules or walls (e.g., yeast, gram-positive bacteria, spores), when workflows prevent processing immediately after collection (e.g., transport from a site of collection to another location for processing), or when samples are numerous (making rapid processing difficult). A common solution to these problems is to freeze the tissue/cells in liquid nitrogen or on dry ice. The frozen samples are often preprocessed to select a desired mass or to partially pulverize the sample before exposure to denaturant. While this freezing and preprocessing allows the researcher more control over the purification conditions, our experience and feedback from customers confirm that this is a complex, time-consuming, and laborious process.

RNAlater® and RNAlater®-ICE RNA Stabilization solutions provide more flexibility and time to allow the researcher to postpone RNA isolation for days, weeks, or even months after tissue collection, without sacrificing the integrity of the RNA. Dissected tissue, body fluids, or collected cells are simply introduced into the RNAlater® solution at room temperature, or into RNAlater®-ICE solution, if frozen. The solution permeates the cells and stabilizes the RNA. The samples are then stored at 4°C using RNAlater® reagent, or at –20°C when using RNAlater®-ICE RNA Stabilization Solution. Samples can be shipped on wet ice or even at room temperature if shipped overnight. Figure 1 shows the integrity of RNA isolated from tissues stored in RNAlater® reagent at 4°C, room temperature, and even at 37°C for increasing lengths of time. Samples stored at 4°C generate intact RNA, even after storage for a month.

Figure 1. Quality of RNA isolated from tissue stored in RNAlater® reagent. Tissues were stored in RNALater® reagent for the indicated times and RNA was purified from the tissues using TRIzol® reagent (Life Technologies™). Equivalent mass amounts of each RNA sample were analyzed using an Agilent® 2100 Bioanalyzer™ instrument. The top panel shows 2100 Bioanalyzer traces of the purified RNA. The bottom panel indicates the yield based on A260 measurement.


Use of RNAlater® solution for tissue storage is compatible with most RNA isolation procedures. Tissues stored in RNAlater® solution are simply removed and processed by homogenization via a Dounce homogenizer, Polytron (Brinkmann), bead disruption, or other mechanical apparatus in the lysis buffer specified by your RNA isolation procedure. Figure 2 shows the RNA isolated from tissue stored in RNAlater® solution using several methods and demonstrates that RNA quality, yield, and signal detection in RT-qPCR by TaqMan® Assay (Life Technologies™) analysis are not affected by storage in RNAlater® solution.


Figure 2. Compatibility of various RNA isolation methods with tissue stored in RNAlater® reagent.  Freshly dissected whole mouse liver and heart were divided and either processed immediately or placed in RNAlater® solution and stored at 4°C for three days prior to processing with TRIzol® reagent or a PureLink® or MagMAX™-96 total RNA kit. Equal mass amounts (X µg) of each purified RNA tissue sample were analyzed by the Agilent® 2100 Bioanalyzer™ instrument. The top panel shows traces of the purified samples from the 2100 Bioanalyzer™ instrument. The bottom panel indicates the yield of each RNA sample based on A260 measurement

Step 2: RNA Preparation

A number of RNA preparation technologies are widely available that can be classified into four general techniques: organic extraction methods, spin basket formats, magnetic particle methods, and direct lysis methods. While all can be used to prepare high-quality RNA suitable for a wide variety of analysis techniques, there are several factors to consider in selecting the right purification technology.

Is the sample particularly difficult to manage?

  • Tissues that are high in nucleases or fatty tissues, and samples with high amounts of inhibitors, can present particular problems.

How much sample do you need to process?

  • Larger sample sizes require kits that contain scalable chemistries. Generally, the larger the sample, the lower the throughput.

What throughput is required?

  • Some formats are particularly well suited to higher levels of throughput and automation.

Organic Extraction Methods

Organic extraction methods are considered the gold standard for RNA preparation. During this process, the sample is homogenized in a phenol-containing solution and the sample is then centrifuged. During centrifugation, the sample separates into three phases: a lower organic phase, a middle phase that contains denatured proteins and gDNA, and an upper aqueous phase that contains RNA. The upper aqueous phase is recovered and RNA is collected by alcohol precipitation and rehydration.

Benefits of organic extraction

  • Rapid denaturation of nucleases and stabilization of RNA
  • Scalable format

Drawbacks of organic extraction

  • The use and associated waste of chlorinated organic reagents
  • Laborious and manually intensive processing
  • Difficult to automate

Filter-based, Spin Basket Formats

Filter-based, spin basket formats utilize membranes (usually glass fiber, derivitized silica, or ion exchange membranes) that are seated at the bottom of a small plastic basket.  Samples are lysed in a buffer that contains RNase inhibitors (usually guanidine salts), and nucleic acids are bound to the membrane by passing the lysate through the membrane using centrifugal force. Wash solutions are subsequently passed through the membrane and discarded. An appropriate elution solution is applied and the sample is collected into a tube by centrifugation. Some formats can be processed by either centrifugation or vacuum using specialized manifolds. Hybrid methods that combine the effectiveness of organic extraction with the ease of sample collection, washing, and elution of spin basket formats also exist.

Benefits of spin basket formats

  • Convenience and ease of use
  • Amenable to single-sample and 96-well processing
  • Ability to automate
  • Ability to manufacture membranes of various dimensions

Drawbacks of spin basket formats

  • Propensity to clog with particulate material
  • Retention of large nucleic acids such as gDNA
  • Fixed binding capacity within a manufactured format
  • When automated, requirements for complex vacuum systems or centrifugation

Magnetic Particle Methods

Magnetic particle methods utilize small (0.5–1 µm) particles that contain a paramagnetic core and surrounding shell modified to bind to entities of interest. Paramagnetic particles migrate when exposed to a magnetic field, but retain minimal magnetic memory once the field is removed. This allows the particles to interact with molecules of interest based on their surface modifications, be collected rapidly using an external magnetic field, and then be resuspended easily once the field is removed. Samples are lysed in a solution containing RNase inhibitors and allowed to bind to magnetic particles. The magnetic particles and associated cargo are collected by applying a magnetic field. After several rounds of release, resuspension in wash solutions, and recapture, the RNA is released into an elution solution and the particles are removed.

Benefits of magnetic particle–mediated purification

  • No risk of filter clogging
  • Solution-based binding kinetics increase the efficiency of target capture
  • The magnetic format allows for rapid collection/concentration of sample
  • Increased ease of implementation on instrument platforms
  • Ability to automate
  • Wide availability of surface chemistries

Drawbacks of magnetic particles

  • Potential carry-through of magnetic particles into eluted samples
  • Slow migration of magnetic particles in viscous solutions
  • Capture/release of particles can be laborious when performed manually

Direct Lysis Methods

Direct lysis methods perform sample preparation (not purification) by utilizing lysis buffer formulations that disrupt samples, stabilize nucleic acids, and are compatible with downstream analysis. Typically, a sample is mixed with lysis agent, incubated for some amount of time under specified conditions, and then used directly for downstream analysis. If desired, samples can often be purified from stabilized lysates. By eliminating the need to bind and elute from solid surfaces, direct lysis methods can avoid bias and recovery efficiency effects that may occur when using other purification methods.

Benefits of direct lysis methods
Extremely fast and easy

  • Highest potential for accurate RNA representation
  • Can work well with very small samples
  • Amenable to simple automation
  • Scalable

Drawbacks of direct lysis methods

  • Inability to perform traditional analytical methods such as spectrophotometric measurement of yield
  • Dilution-based (most useful with concentrated samples)
  • Potential for suboptimal performance unless developed/optimized with downstream analysis
  • Potential for residual RNase activity if lysates are not handled properly

Ambion® RNA isolation kits provides flexibility for sample size, type, and processing format, and includes kits for the isolation of total or poly(A) RNA. For additional information on approximately how much total or poly(A) RNA can be recovered from a given amount of tissue or cells, please refer to the technical information that accompanies each kit, or contact Life Technologies Technical Support.

Step 3: Quantitation of Isolated RNA

RNA quantitation is an important and necessary step prior to most RNA analysis methods. Here we discuss three common methods used to quantitate RNA and tips for optimizing each of these methods.

UV Spectroscopy

The traditional method for assessing RNA concentration and purity is UV spectroscopy. The absorbance of a diluted RNA sample is measured at 260 and 280 nm. The nucleic acid concentration is calculated using the Beer-Lambert law, which predicts a linear change in absorbance with concentration (Figure 3).

 

 

Figure 3. Beer-Lambert Law for calculating UV absorbance by nucleic acid.

Using this equation, an A260 reading of 1.0 is equivalent to ~40 µg/mL single-stranded RNA. The A260/A280 ratio is used to assess RNA purity. An A260/A280 ratio of 1.8–2.1 indicates highly purified RNA. UV spectroscopy is the most widely used method to quantitate RNA. It is simple to perform, and UV spectrophotometers are available in most laboratories. The method does have several drawbacks, but they can be minimized by following these tips:

  • This method does not discriminate between RNA and DNA, and it is advisable to first treat RNA samples with RNase-free DNase to remove contaminating DNA.
  • Other contaminants such as residual proteins and phenol can interfere with absorbance readings, so care must be taken during RNA purification to remove them.
  • Sample readings are made in quartz cuvettes. Dirty cuvettes and dust particles cause light scatter at 320 nm, which can impact absorbance at 260 nm. Since neither proteins nor nucleic acids absorb at 320 nm, perform a background correction by making readings from a blank (diluent only) at 320 nm, as well as 260 nm and 280 nm.
  • The A260/A280 ratio is dependent on both pH and ionic strength. As pH increases, the A280 decreases while the A260 is unaffected. This results in an increasing A260/A280 ratio [1]. Because water often has an acidic pH, it can lower the A260/A280 ratio. We recommend using a buffered solution with a slightly alkaline pH, such as TE (pH 8.0), as a diluent (and as a blank) to assure accurate and reproducible readings. An example of the variation in A260/A280 ratio at different pH values is shown in Figure 4.
  • Make sure your RNA dilution is within the linear range of your spectrophotometer. Usually absorbance readings should fall between 0.1 and 1.0. Solutions that give readings outside this range cannot be measured accurately. Generally the greatest error occurs at lower concentrations.

Figure 4. Effects of pH on A260/A280 ratio.

Fluorescent Dyes

Certain fluorescent dyes, such as the Quant-iT™ RiboGreen® RNA Reagent, exhibit a large fluorescence enhancement when bound to nucleic acids. As little as 1 ng/mL of RNA can be detected and quantified using the RiboGreen® reagent with a standard fluorometer, fluorescence microplate reader, or filter fluorometer. To accurately quantitate RNA, unknowns are plotted against a standard curve produced with a sample of known concentration, usually based on its absorbance at 260 nm. The linear range of quantitation with RiboGreen® reagent can extend three orders of magnitude (1 ng/mL to 1 µg/mL) when two different dye concentrations are used. Furthermore, Quant-iT™ RiboGreen® RNA Reagent assays are relatively insensitive to non–nucleic acid contaminants commonly found in nucleic acid preparations, so that linearity is maintained. This method of quantifying RNA can be optimized using the following tips:

  • In addition to RNA, Quant-iT™ RiboGreen® RNA Reagent can bind to DNA and fluoresce. It is advisable to treat RNA samples with RNase-free DNase to remove contaminating DNA prior to quantitation.
  • The Quant-iT™ RiboGreen® RNA Reagent can adsorb to the sides of tubes. This can be minimized by preparing solutions in nonstick nuclease-free polypropylene plasticware.
  • Protect the RiboGreen® RNA Reagent from photodegradation by wrapping the container with foil, and use the reagent within several hours of preparation.
  • Avoid repeated freeze-thaw cycles of RNA standards. This can cause strand-scission of the RNA, resulting in decreased dye binding. In addition, nucleic acids can absorb to tubes with repeated freeze-thaw cycles. This phenomenon becomes more pronounced at lower concentrations.

Agilent® 2100 Bioanalyzer™ Instrument

The Agilent® 2100 Bioanalyzer™ instrument uses a combination of microfluidics, capillary electrophoresis, and fluorescent dye that binds to nucleic acid to evaluate both RNA concentration and integrity. After priming the Bioanalyzer™ Lab Chip with separation matrix, RNA ladder and samples are loaded in designated wells on the chip. Size and mass information is provided by the fluorescence of RNA molecules as they move through the channels of the chip. The instrument software automatically compares the peak areas from unknown RNA samples to the combined area of the six Agilent® RNA 6000 Ladder RNA peaks to determine the concentration of the unknown samples. The Agilent® RNA 6000 Nano System has a broad dynamic range and can quantify 25–500 ng/µL of RNA, while the Agilent® RNA 6000 Pico Chip System can quantify 50–5,000 pg/µL of RNA.

Perhaps the most powerful feature of the Agilent® 2100 Bioanalyzer™ instrument is its ability to provide information about RNA integrity. As each RNA sample is analyzed, the software generates both a gel-like image and an electropherogram. When analyzing total RNA, an analysis algorithm is used to assess the integrity of the RNA sample (the RNA Integrity Number, or RIN) with a maximum value of 10. Significant decreases in the RIN are indicative of degraded total RNA.

Step 4: Storage of Isolated RNA

The last step in every RNA isolation protocol, whether for total or mRNA preparation, is to resuspend the purified RNA pellet. After painstakingly preparing an RNA sample, it is crucial that RNA be suspended and stored in a safe, RNase-free environment. We recommend storing RNA at –80°C in single-use aliquots, resuspended in one of several RNA storage solutions designed for this purpose:

  • THE RNA Storage Solution (1 mM sodium citrate, pH 6.4 ± 0.2)
  • 0.1 mM EDTA (in DEPC-treated ultrapure water)
  • TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0)
  • RNAsecure™ Resuspension Solution

TE and 0.1 mM EDTA solutions are often specified in common RNA isolation and analysis protocols. These storage solutions are ideal for researchers who already use them, but would like the convenience and security of having them premade and certified RNase-free.

We also have introduced THE RNA Storage Solution, a buffer that delivers greater RNA stability than 0.1 mM EDTA or TE. THE RNA Storage Solution has two features that minimize base hydrolysis of RNA: low pH, and sodium citrate, which acts both as a pH buffer and a chelating agent (divalent cations catalyze base hydrolysis of RNA). THE RNA Storage Solution is compatible with all of the common RNA applications such as reverse transcription, in vitro translation, northern analysis, and nuclease protection assays.

The RNAsecure™ Reagent is a unique nonenzymatic reagent for the irreversible inactivation of RNases in enzymatic reactions. It is supplied at a 25X concentration and can be added to samples to inactivate RNases. RNAsecure™ Resuspension Solution contains the same active ingredients as the RNAsecure™ Reagent, but is supplied at a working concentration for direct resuspension of RNA pellets. To inactivate RNases, the RNA pellet is resuspended in the RNAsecure™ Resuspension Solution and heated to 60°C for 10 minutes. A unique feature of the RNAsecure™ solution is that reheating after the initial treatment will reactivate the RNase-destroying agent to minimize any new contaminants.

Life Technologies is continuously inventing ways to make RNA analysis easier. We work closely with our customers and colleagues to provide unique products to solve the problems researchers frequently face when working with RNA. Ambion® technology underlies RNAlater® Solution, RNA isolation kits, and RNA storage solutions. Ambion® RNA analysis solutions are designed to work together to take you all the way from sample collection to your RNA analysis application. If you have suggestions for additional products that would be useful in your RNA research, please contact us.

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References

1. Wilfinger WW, Mackey K, and Chomczynski P (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22:474­481.

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For Research Use Only. Not intended for human or animal therapeutic or diagnostic use.