What is RNA?
RNA is a biological macromolecule that serves a number of different functions. Messenger RNA (mRNA), transcribed from DNA, serves as a template for synthesis of proteins. Protein synthesis is carried out by ribosomes, which consist of ribosomal RNA (rRNA) and proteins. Amino acids for protein synthesis are delivered to the ribosome on transfer RNA (tRNA) molecules. RNAs are also part of riboproteins involved in RNA processing.
Noncoding RNAs are also important. These are functional RNA molecules that do not translate into proteins. Such RNAs include tRNA and rRNA, as well as small nucleolar RNAs (snoRNA), microRNAs (miRNA), short interfering RNAs (siRNA) and piwi-interacting RNAs (piRNA). They are often involved in the regulation of gene expression.
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microRNA
microRNAs (miRNAs) are a class of endogenous (naturally occurring), ~22 nucleotide, noncoding RNAs that mediate post-transcriptional gene regulation (see video miRNA biogenesis and mode of action). They exhibit similar characteristics to siRNAs.
miRNAs play an important role in many biological processes, including differentiation and development, cell signaling, and response to infection. Overwhelming evidence indicates that dysregulation of miRNA expression is a cause or indicator of several disease processes, including many cancers. The discovery that cell-free miRNAs are detectable in serum and plasma, and that their expression varies as a result of disease, presents great potential for cell-free miRNA expression signatures to be used as biomarkers in disease diagnosis and prevention.
Both miRNA and siRNA pathways involve double-stranded RNA, but the source of these RNAs differs. Unlike the double-stranded RNA that triggers RNAi, miRNAs are encoded in the genome. Additionally, miRNA precursors (pre-miRNAs) are not completely double-stranded, but rather form hairpin-like structures that contain double-stranded regions. In contrast to RNAi, the miRNA pathway focuses on regulating the cell’s own genes. It is believed that humans have more than 2000 miRNAs, and they are estimated to regulate as over two-thirds of human genes.
The miRNA system is an endogenous mechanism of regulation of gene expression. Mature miRNAs contribute to the regulation of endogenous genes, primarily by translational repression. In addition, miRNAs can mediate mRNA destruction by rapid deadenylation and/or decapping. Naturally occurring miRNA-binding sites are typically found in the 3' untranslated regions (UTRs) of target mRNAs. In animal miRNA, partial complementarity has made positive identification of true binding sites difficult and imprecise.
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miRNA mimics and inhibitors
miRNA mimics are chemically synthesized, double-stranded RNAs (usually 18–24 nucleotides) which mimic mature endogenous miRNAs after transfection into cells. miRNA inhibitors are single-stranded (usually 21–25 nucleotides), modified RNAs which, after transfection, specifically inhibit miRNA function. Mimics and inhibitors may also contain chemical modifications proposed to improve activity or in vivo stability.
Transfection of mimics, followed by downstream gene expression analysis or phenotypic analysis, is performed to elucidate the targets and roles of particular miRNAs. These experiments enable study of the biological effects of misregulation of individual miRNAs, as well as confirmation of specific genes as targets of individual miRNAs. Reduced gene expression after transfection of an miRNA mimic or increased expression after transfection of an miRNA inhibitor provides evidence that the miRNA under study is involved in regulation of that gene. Alternatively, the role of miRNAs in various pathways can be studied by examination of a specific phenotype following miRNA mimic or inhibitor transfection.
Downstream analysis of the effect of miRNA mimic/inhibitor transfection is often performed using one of the following strategies:
- A plasmid vector which carries a reporter gene such as luciferase and one or more miRNA binding sites in the 3' UTR is used as an miRNA target. miRNA mimic and/or inhibitor is cotransfected with the vector. After transfection, a reporter assay, such as a luciferase assay, is performed. The effect of the mimic/inhibitor is determined by comparing this to the result from cells transfected with the vector alone.
- The expression of an endogenous gene, which is known to be a target of the miRNA under study, is measured after mimic/inhibitor transfection. The effect of the mimic/inhibitor is determined by comparing this result with the gene expression in untransfected cells or cells transfected with a negative control. Gene expression is often measured at the protein level, for example, by western blot, as miRNAs often inhibit translation of their target genes and do not cause degradation of the target transcript. This means that the effect of an miRNA mimic or inhibitor can often not be determined using quantitative, real-time PCR.
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siRNA and RNAi
siRNA
Small interfering RNA (more commonly known as siRNA) is involved in a number of biological processes — most commonly RNA interference, or RNAi.
Most RNA is single stranded; however, siRNA is made up of two complementary strands of nucleotides, similar to those in DNA, and is about 20–25 nucleotides in length. siRNA plays a role in the RNAi pathway, where it interferes with the expression of specific genes with complementary nucleotide sequence.
Approximate values for a double-stranded, 21 nt siRNA molecule:
- 20 µM siRNA is equivalent to approximately 0.25 µg/µl
- Molecular weight of a 21 nt siRNA is approximately 13–15 µg/nmol (sequence-dependent)
RNAi
RNA interference (or RNAi) is a natural cellular process whereby cells 'turn down,' or silence, the activity of specific genes. Discovered in 1998, it is now a powerful tool in the study of gene function.
RNAi works by disrupting the messages carried by messenger RNA (mRNA) and therefore suppressing protein synthesis. Without the mRNA activity, a gene is essentially inactive.
Upon entering a cell, the double-stranded RNA molecules (siRNA) that trigger RNAi are cleaved into small fragments by an enzyme called Dicer. The small fragments then serve as guides, leading the siRNA to mRNAs that match the genetic sequence of the fragments. These cellular mRNAs are then cleaved, effectively destroying their messages and silencing the corresponding gene.
The process of RNAi is complex (see figure The process of RNAi). Double-stranded RNA is recognized by an RNase III and cleaved into siRNAs of 21–23 nucleotides. These siRNAs are incorporated into an RNAi targeting complex known as RISC (RNA-induced silencing complex), which destroys mRNAs homologous to the integral siRNA. The target mRNA is cleaved in the center of the region complementary to the siRNA resulting in degradation of the target mRNA and decreased protein expression.
Using siRNA
siRNAs are the main effectors in RNAi and can be synthesized chemically or enzymatically in vitro. The design of an siRNA sequence is critical to effective gene silencing and designs are developed based on an understanding of RNAi and the naturally occurring siRNA function.
siRNA delivery is critical in such gene silencing experiments. Synthetic siRNAs can be delivered by electroporation or by using lipophilic agents. However, both approaches are transient. Plasmid systems can also be used to express small hairpin RNAs (shRNAs) that are substrates for Dicer and mature into siRNAs in vivo. Such systems enable stable suppression of target genes. Various viral delivery systems have also been developed to deliver shRNA into difficult-to-transfect cell lines.
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General remarks on RNA handling, storage and stabilization
Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function. Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA, do not use any plasticware or glassware without first eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure. In order to create and maintain an RNase-free environment, precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA.
In order to ensure accurate gene expression analyses, it is important that the RNA analyzed truly represents the in vivo gene expression of the sample. This is complicated by the fact that changes can occur during handling of the sample and isolation of the RNA.
Once a biological sample is harvested, its RNA becomes extremely unstable. There are two major types of artifacts that can occur. Downregulation of genes and enzymatic degradation of RNA result in an artificial reduction of both nonspecific and specific mRNA species. At the same time, certain genes can be induced during handling and processing of the sample. The combination of these two effects can result in a transcription profile that differs from the true in vivo gene expression pattern. Immediate stabilization of the RNA expression pattern is a prerequisite for accurate gene expression analysis.
In addition, purified RNA stored under proper temperature conditions show no sign of degradation even after 1 year.
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RNA isolation: Disruption and homogenization of starting materials
Efficient disruption and homogenization of the starting material is an absolute requirement for all total RNA isolation procedures. Disruption and homogenization are two distinct steps.
- Disruption: Complete disruption of tissue structure, cell walls, and plasma membranes of cells is absolutely required to release all the RNA contained in the sample. Different samples require different methods to achieve complete disruption. Incomplete disruption results in significantly reduced yields.
- Homogenization: Homogenization is necessary to reduce the viscosity of the cell lysates produced by disruption. Homogenization shears the high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate. Incomplete homogenization results in inefficient binding of RNA and therefore significantly reduced yields.
Some disruption methods simultaneously homogenize the sample while others require an additional homogenization step. The infographic gives an overview of different disruption and homogenization methods suitable for various starting materials. It can be used as a guide to choose the appropriate method for the starting material with which you are working. The disruption and homogenization methods are described in more detail below.
Disruption and homogenization using bead mills
In disruption using a bead mill, the sample is agitated at high speed in the presence of beads. Disruption and simultaneous homogenization occur by the hydrodynamic shearing and crushing action of the beads as they collide with the cells. Disruption efficiency is influenced by:
- Size and composition of beads
- Ratio of buffer to beads
- Amount of starting material
- Speed and configuration of agitator
- Disintegration time
The optimal beads to use are 0.1 mm (mean diameter) glass beads for bacteria, 0.5 mm glass beads for yeast and unicellular animal cells, and 3–7 mm stainless steel beads for animal and plant tissues. It is essential that glass beads are pretreated by washing in concentrated nitric acid. Alternatively, use commercially available acid-washed glass beads. All other disruption parameters must be determined empirically for each application. Plant material as well as the beads and disruption vessels can be precooled in liquid nitrogen, and disruption should be performed without lysis buffer. Dry, cryogenic grinding is also used for animal tissue. Cryogenic grinding (regardless of whether in a bead mill or by mortar and pestle) does not homogenize the sample, unlike when lysis buffer is used.
Disruption and homogenization using rotor–stator homogenizers
Rotor–stator homogenizers thoroughly disrupt and simultaneously homogenize, in the presence of lysis buffer, animal tissues in 5–90 seconds depending on the toughness of the sample. Rotor–stator homogenizers can also be used to homogenize cell lysates. The rotor turns at a very high speed causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing. Foaming of the sample should be kept to a minimum by using properly sized vessels, by keeping the tip of the homogenizer submerged, and by holding the immersed tip to one side of the tube. Rotor–stator homogenizers are available in different sizes and operate with differently sized probes. Probes with diameters of 5 mm and 7 mm are suitable for volumes up to 300 µl and can be used for homogenization in microfuge tubes. Probes with a diameter of 10 mm or above require larger tubes.
Disruption using a mortar and pestle
For disruption using a mortar and pestle, freeze the sample immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen. Transfer the suspension (tissue powder and liquid nitrogen) into a liquid-nitrogen-cooled, appropriately sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw. Add lysis buffer and continue as quickly as possible with the procedure.
Note: Grinding the sample using a mortar and pestle will disrupt the sample, but it will not homogenize it. Homogenization must be performed separately before proceeding.
Homogenization using a syringe and needle
Cell and tissue lysates can be homogenized using a syringe and needle. High-molecular-weight DNA can be sheared by passing the lysate through a 20-gauge (0.9 mm) needle, attached to a sterile plastic syringe, at least 5–10 times or until a homogeneous lysate is achieved. Increasing the volume of lysis buffer may be required to facilitate handling and minimize sample loss.
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Special considerations for isolation of RNA from different sample sources
Some sample sources have differences in their RNA or contain substances that can cause problems in RNA isolation and analysis. Special considerations are required when working with these sample sources. In this infographic, considerations for working with a number of different sources are discussed.
No currently available purification method can guarantee that RNA is completely free of DNA, even when it is not visible on an agarose gel. Certain solutions that can help remove genomic DNA contamination from RNA are also highlighted in this infographic.
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RNA numbers: Size, molecular weight, distribution, yield, and conversions
A typical rapidly growing mammalian cell culture contains 10–30 pg total RNA per cell, whereas a fully differentiated primary cell will contain far less — in the region of <1 pg per cell. The majority of RNA molecules are tRNAs and rRNAs. mRNA accounts for only 1–5% of the total cellular RNA although the actual amount depends on the cell type and physiological state. Approximately 360,000 mRNA molecules are present in a single mammalian cell, made up of approximately 12,000 different transcripts with a typical length of approximately 2 kb. Some mRNAs comprise as much as 3% of the mRNA pool whereas others account for less than 0.01%. These “rare” or “low abundance” messages may have a copy number of only 5–15 molecules per cell. However, these rare species may account for as much as 11,000 different mRNA species, comprising 45% of the mRNA population. All such important RNA numbers are represented in this infographic.
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Quantification and analysis of RNA
Depending on the downstream application, four main quality parameters can characterize the quality of your RNA samples – quantity, purity, size and sequence. Variations in these parameters can impact your experiments and affect the data quality and results interpretation. Even small variations can mean the difference between assay success or failure.
Quantity
Molecular biology reactions require very precise amounts of RNA for optimal performance. Too little or too much RNA can severely impact the final assay results, so quantification should be a standard procedure following purification to ensure successful reaction outcomes.
Purity
RNA samples can become contaminated by other molecules with which they were co-extracted and eluted during the purification process or by chemicals from upstream applications. The resulting impurities can significantly decrease the sensitivity and efficiency of your downstream enzymatic reactions. Along with identifying common chemical contaminants, differentiating between different types of nucleic acids (RNA vs. DNA) is also important when assessing sample purity.
Size and integrity
Upstream processing or contaminating nucleases can lead to degradation or fragmentation, and problems with enzymatic reactions and biochemical modifications may result in unexpected fragment sizes. Therefore, it is important to check the condition of your RNA samples prior to analysis.
Sequence
Sequencing a sample is the ultimate QC step scientists should perform to verify and validate that the pieces of RNA they are working with are the correct ones and that the genetic information has not been altered in any way along the workflow.
In this infographic, we describe the influence of these parameters on downstream applications, how to assess them and how to implement quick and simple quality control measures that can help increase your RNA research success.
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References
- Alberts, B., et al. (2002) Molecular Biology of the Cell. 4th ed. New York: Garland Publishing, Inc.
- Ausubel, F.M. et al. (1991) Current Protocols in Molecular Biology, New York: John Wiley and Sons.
- Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.
- Sambrook, J., Fritsch, E.F., and Maniatis, T. (2012) Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
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