Our guide is designed to steer you towards the successful and dependable isolation of ready-to-use RNA, catering to a diverse range of downstream experiments, including RNA-seq and cDNA generation. Ensuring the purity of RNA, especially in the case of precious and rare samples, requires meticulous purification and decontamination procedures.
A Beginner's Guide to RNA Sequencing may be a helpful article for you to plan your next RNA sequencing project.
RNA, being a highly labile substance, demands careful handling. Given its single-stranded structure and the omnipresence of RNase in the laboratory environment, exercising extreme caution during the RNA extraction process is imperative. Operating within an RNase-free environment throughout the entire procedure is crucial, necessitating the use of appropriate protective gear such as masks and gloves.
Principles of RNA Extraction
The fundamental approach to RNA extraction revolves around the utilization of single-phase lysis reagents. This method involves lysing the biological sample through disruption with a single-phase solution containing guanidine isothiocyanate and phenol. Subsequently, chloroform (or bromochloropropane) is introduced, creating a mixture that is then centrifuged. This centrifugation process results in the separation of the homogenate into distinct aqueous and organic phases. Notably, RNA is selectively present in the upper aqueous phase, DNA is located at the boundary between the aqueous and organic phases, and denatured proteins are found in the lower organic phase.
Following the recovery of the aqueous phase, RNA is precipitated by introducing isopropanol. For cases where both DNA and protein extraction are necessary, a sequential precipitation approach is employed. The intermediate phase, containing DNA, is precipitated with ethanol, while the protein is precipitated with isopropanol from the organic phase.
The DNA obtained from the intermediate phase is approximately 20kb in size and serves as a suitable template for Polymerase Chain Reaction (PCR). Notably, the proteins, having undergone denaturation due to exposure to guanidine salts, are primarily utilized for immunoblotting applications.
RNA extraction from blood samples is efficiently facilitated by utilizing the PAXgene Whole Blood RNA Blood Collection Tube. This specialized tube overcomes the inherent challenges associated with blood RNA, known for its susceptibility to degradation. The proprietary reagents within the blood tubes play a pivotal role in stabilizing RNA, ensuring the production of gene expression data that faithfully reflects the precise state of the blood at the moment of sampling.
If you are interested in Blood RNA Sequencing Service, you can refer to our RNA Sequencing Sample Submission and Preparation Guidelines.
Workflow of RNA and DNA purification. (Yamagata et al., 2021)
Common RNA Extraction Methods
Laboratories commonly employ various methods for extracting RNA from plant and animal tissues as well as cells. One traditional approach involves the use of RNA extraction reagents that require the addition of chloroform. It's worth noting that chloroform is a highly toxic and volatile hazardous chemical. In response to safety concerns, modern RNA extraction reagents are available, and many of them do not contain chloroform.
Among the diverse RNA extraction methods, a widely utilized non-kit method is the guanidino acid phenol extraction, commonly known as TRIzol and TRIreagent. This approach offers an effective means of RNA extraction, suitable for different types of samples. Importantly, it eliminates the need for chloroform, addressing safety considerations associated with its use.
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Our TRIzol RNA Extraction Protocol may be helpful for you.
Table 1 Common RNA Extraction Methods
| RNA Extraction Method |
Description |
| Guanidino Acid Phenol Extraction |
Utilizes liquid-liquid extraction with centrifugation. RNA is retained in the upper aqueous phase, while proteins and DNA separate into the lower phenol phase. RNA is precipitated using alcohol. |
| Silica Technology, Glass Fiber Filters |
Solid-phase extraction typically in the form of column spin filters used in commercial kits. RNA binds to silica (dependent on salt dissociation), is washed, and then eluted. |
| Cesium Chloride/Trifluoroacetic Acid |
Liquid-liquid extraction method employing CsCl gradient generated by high-speed centrifugation. RNA is precipitated, washed, re-suspended, and further precipitated with alcohol. |
| Magnetic Bead Technology |
Utilizes highly superparamagnetic 20-30 nm particles with different surface materials (e.g., silica) capable of capturing RNA. Particles are captured using an external magnetic field through washing and elution steps. |
| Lithium Chloride and Urea Separation |
Tissues homogenized in urea lithium to precipitate RNA. Protein is separated from RNA using a phenol-chloroform solution. RNA is then precipitated with ethanol. |
| Lithium Chloride (LiCl) Precipitation |
LiCl serves as an alternative to alcohol precipitation and is advantageous for RNA extraction as it preferentially precipitates RNA over DNA. |
| Oligo(dT)-Cellulose Column |
Useful column chromatography method capturing RNA on oligo dT-cellulose resin through complementary base pairing, thus separating mRNA or synthetic RNA with Poly(A) tails. |
Choosing the Right RNA Separation Method
Choosing the right RNA isolation method for RNA sequencing involves considering the sample type, RNA type (e.g., miRNA, tRNA), the quantity needed for downstream applications, and the number of samples to process. Assess the efficiency of lysis, compatibility of re-suspension solutions with sequencing applications, and the potential need to isolate other molecules (e.g., DNA, proteins). Safety, cost, and convenience are also important factors. Reviewing published protocols and validating the chosen method for specific sample types and downstream applications ensures the reliability of RNA for successful RNA sequencing experiments.
CD Genomics provides a range of tailored guides for sample handling and submission, along with RNA sequencing services tailored to diverse projects and sample types. Explore our comprehensive RNA Sequencing Sample Submission and Preparation Guide for detailed information on the process and requirements.
In our Guideline, we accommodate a diverse range of samples and provide comprehensive processing methods, including but not limited to animal tissues, cell samples, plant tissues, bodily fluid samples, microbial samples, and extracellular vesicle samples.
Quantifying RNA and Assessing Quality
Before using RNA, it's crucial to determine its quantity and quality. UV spectroscopy is commonly used for quantification, measuring A230, A260, and A280. The RNA concentration (μg/mL) can be calculated using the formula: RNA sample concentration = A260 × 40 × dilution. To address DNA contamination, treat RNA samples with RNase-free DNase.
Contaminants like phenol and proteins can affect readings. The A260/A280 ratio assesses protein contamination, with pure RNA having a ratio close to 2.0. The A260/A230 ratio evaluates contamination by organic compounds or salts, ideally close to 2.0 for RNA.
The A260/A280 ratio is pH-sensitive. Using a buffer like TE (pH 8.0) for dilution and a blank control ensures accurate and reproducible readings. Keep in mind that the method doesn't distinguish between RNA and DNA.
1.0 of A260 corresponds to approximately 40 µg/mL single-stranded RNA (assuming pH ~7.5). An A260/A280 ratio of 1.8-2.1 indicates a high degree of RNA purification.
Treat samples with RNase-free DNase for accurate results.
- Agarose Gel Electrophoresis Analysis
To assess the quality of extracted RNA, perform non-denaturing agarose gel electrophoresis using MOPS or TBE buffer. Prepare a 0.8% agarose gel and run electrophoresis at a voltage of 4~5 V/cm. For higher purity RNA, expect clear and distinct three (four) bands representing 28S, 18S, and 5.8/5S rRNA. A uniform brightness across the bands indicates consistent RNA quality in the upper sample. This simple technique provides visual confirmation of RNA integrity, aiding in the evaluation of RNA suitability for downstream applications.
Troubleshooting Guide: RNA Extraction for Sequencing may be a helpful article.
18S rRNA and 28S rRNA electrophoresis bands for RNA samples extracted from samples. (Aboutalebi et al., 2020)
RNA Quality Control
For subsequent experiments, particularly RNA sequencing, the success or failure of sequencing is directly impacted by the RNA quality control process. It is paramount to evaluate RNA integrity and purity, achievable through the measurement of the 28S:18S ribosomal RNA (rRNA) ratio. Higher RIN values generally signify superior RNA integrity, making them suitable for downstream analyses. However, formalin-fixed paraffin-embedded (FFPE) tissues are notorious for harboring low-quality RNA, typically reflected in RIN values ranging from 2 to 5. Unfortunately, there are currently no dependable methods to predict the success of RNA sequencing experiments based on FFPE tissues. Success assessment relies on various factors such as storage time, conditions, fixation duration, and sample size.
Please read our article: RNA Sequencing Quality Control for more information.
References:
-
Yamagata, Hirotaka, et al. "Optimized protocol for the extraction of RNA and DNA from frozen whole blood sample stored in a single EDTA tube." Scientific reports 11.1 (2021): 17075.
- Aboutalebi, Elmira, et al. "Investigating the effect of Sclareol on IRE-1 and PERK genes the pathway of reticulandaplasmic system stress in gastric cancer cells MKN-45." Journal of Research in Applied and Basic Medical Sciences 6.1 (2020): 44-32.
