RNA Protocols & Applications

Protocols and applications for RNA, including RNAi, siRNA, miRNA, mimics, and inhibitors
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This section describes considerations for isolation and quantification of RNA from different sample sources and RNA storage. It also deals with RNAi and the use of siRNA, together with miRNA, mimics, and inhibitors.
What is RNA?
microRNA
miRNA mimics and inhibitors
siRNA and RNAi
General remarks on handling RNA
Stabilization of RNA in biological samples
RNA sizes and molecular weights
RNA isolation: Disruption and homogenization of starting materials
Special considerations for isolation of RNA from different sample sources
Storage of RNA
Quantification of RNA
Spectrophotometric measurement of RNA concentration
Determining RNA quality
RNA analysis: Analytical gels
Running and analyzing formaldehyde gels for RNA analysis
RNA analysis: Northern blotting
Capillary electrophoresis
RNA integrity parameters
Removal of genomic DNA contamination from RNA
References

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.

miRNAs are a class of endogenous (naturally occurring), ~18–24 nucleotide, noncoding RNAs that mediate post-transcriptional gene regulation. Individual miRNAs can be present in excess of 105 copies per cell, but are negligible in terms of overall RNA mass per cell. Due to their short length, they usually require specialized isolation and analysis protocols.

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 (see tables RNA content of a typical human cell and RNA distribution in a typical mammalian cell). 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 (see tables mRNA classification based on abundance and RNA content in various cells and tissues and reference 1 for more information).

While the genes of an organism are relatively fixed, the mRNA population represents how genes are expressed under any given set of conditions. Analysis of RNA by hybridization technologies, including northern blotting and microarray analysis, or by RT-PCR, or by transcriptome sequencing (RNA-seq) can provide a good reflection of an organism’s gene expression profile. Compared to DNA, however, RNA is relatively unstable. This is largely due to the presence of ribonucleases (RNases), which break down RNA molecules.

RNases are very stable, do not require cofactors, are effective in very small quantities, and are difficult to inactivate. RNase contamination can come from human skin and dust particles, which can carry bacteria and molds. Isolation and analysis of RNA therefore requires specialized techniques.

This section describes procedures for successful stabilization, purification, and analysis of RNA.

RNA content of a typical human cell
Parameter Amount
 Total RNA per cell  <1–30 pg
 Proportion of total RNA in nucleus  ~14%
 DNA:RNA in nucleus  ~2:1
 mRNA molecules  2 x 105 – 1 x 106
 Typical mRNA size  1900 nt

RNA distribution in a typical mammalian cell
RNA species Relative amount
 rRNA (28S, 18S, 5S)  80–85%
 tRNAs, snRNAs, low MW species  15–20%
 mRNAs  1–5%
 

mRNA classification based on abundance
Abundance  Copies/cell  Number of different messages per cell Abundance of each message
 Low  5–15  11,000  <0.004%
 Intermediate  200–400  500  <0.1%
 High  12,000  <10  3%

RNA content in various cells and tissues
Organism  Source Total RNA (µg)*
 Cell cultures (1 x 106 cells)  NIH/3T3
 HeLa 
 COS-7
 LMH
 Huh
 10
 15
 35
 12
 15
 Mouse/rat tissues (10 mg)  Embryo (13-day)
 Kidney
 Liver
 Spleen
 Thymus
 Lung
 25
 20–30
 40–60
 30–40
 40–50
 10–20
 Yeast (1 x 107 cells)  S. cerevisiae  25
 Plant (100 mg leaves)  Arabidopsis
 Maize
 Tomato
 Tobacco
 35
 25
 65
 60
* Amounts can vary due to factors such as species, developmental stage, and growth conditions.

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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 figure The canonical pathway of miRNA biogenesis). They exhibit similar characteristics to siRNAs (see siRNA).

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 (see 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 (see 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 handling RNA

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, the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA.

General handling

Proper microbiological, aseptic technique should always be used when working with RNA. Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination. Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment. Change gloves frequently and keep tubes closed whenever possible. Keep purified RNA on ice when aliquots are pipetted for downstream applications.

To remove RNase contamination from bench surfaces, nondisposable plasticware, and laboratory equipment (e.g., pipets and electrophoresis tanks), a commercial RNase removal solution is recommended. RNase contamination can alternatively be removed using general laboratory reagents. To decontaminate plasticware, rinse with 0.1 M NaOH, 1 mM EDTA followed by RNase-free water (see Solutions), or rinse with chloroform if the plasticware is chloroform-resistant. To decontaminate electrophoresis tanks, clean with detergent (e.g., 0.5% SDS), rinse with RNase-free water, rinse with ethanol (if the tanks are ethanol resistant), and allow to dry.

Important: When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.

Disposable plasticware

The use of sterile, disposable polypropylene tubes is recommended when working with RNA. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases.

Non-disposable plasticware

Non-disposable plasticware should be treated before use to ensure that it is RNase-free. Plasticware should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA followed by RNase-free water (see Solutions). Alternatively, chloroform-resistant plasticware can be rinsed with chloroform to inactivate RNases.

Glassware

Glassware should be treated before use to ensure that it is RNase-free. Glassware used for RNA work should be cleaned with a detergent, thoroughly rinsed, and oven baked at 240°C for at least 4 hours (overnight, if more convenient) before use. Autoclaving alone will not fully inactivate many RNases. Alternatively, glassware can be treated with DEPC (diethyl pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water), incubate overnight (12 hours) at 37°C, and then autoclave or heat to 100°C for 15 minutes to eliminate residual DEPC.

Important: When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.

Electrophoresis tanks

Electrophoresis tanks should be cleaned with detergent solution (e.g., 0.5% SDS), thoroughly rinsed with RNase-free water, and then rinsed with ethanol and allowed to dry.  

Important: When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.

Important: Plastics used for some electrophoresis tanks are not resistant to ethanol. Take proper care and check the supplier’s instructions.

Solutions

Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong, but not absolute, inhibitor of RNases that works by covalently modifying RNases.

Important: When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.

Preparation of RNase-free solutions

Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of 0.1% to inactivate RNases on glass or plasticware or to create RNase-free solutions and water. DEPC inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for 12 hours at 37°C. Autoclave for 15 minutes to remove any trace of DEPC. DEPC will react with primary amines and cannot be used directly to treat Tris buffers. DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO2. When preparing Tris buffers, treat water with DEPC first, and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation. Carbethoxylated RNA is translated with very low efficiency in cell-free systems. However, its ability to form DNA:RNA or RNA:RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified. Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100°C for 15 minutes.

Important: When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.

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Stabilization of RNA in biological samples

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. Traditionally, samples harvested for RNA analysis are immediately frozen in liquid nitrogen and stored at –80°C until processed. Stabilization reagents, available from commercial suppliers, can alternatively be used to stabilize RNA in biological samples. Integrated sample handling solutions, including containers, stabilization reagents, and preparation kits are available from commercial suppliers.

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RNA sizes and molecular weights 

Sizes and molecular weights of various RNAs
RNA  Nucleotides Molecular weight (daltons)
E. coli
  tRNA
  5S rRNA
  16S rRNA
  23S rRNA

 75
 120
 1541
 2904

 2.6 x 104
 4.1 x 104
 5.2 x 105
 9.9 x 105
Drosophila
  18S rRNA
  28S rRNA
 
 1976
 3898
 
 6.7 x 105
 1.3 x 106
Mouse
  18S rRNA
  28S rRNA
 
 1869
 4712
 
 6.4 x 105
 1.6 x 106
Rabbit
  18S rRNA
  28S rRNA
 
 2366
 6333
 
 8.0 x 105
 2.2 x 106
Human
  18S rRNA
  28S rRNA
 
 1868
 5025

 6.4 x 105
 1.7 x 106
Adapted from reference 2.

Conversions for nucleic acids: RNA

Molecular weight and molar conversions for RNA

  • MW of a single-stranded RNA molecule (sodium salt) = (number of bases) x (343 daltons/base) 

Molar conversions for RNA*
Micrograms of RNA  Picomoles Molecules
 1.0  1.67  1.0 x 1012
 0.6  1.0  1.0 x 1011
* Average mRNA 1930 nucleotides in length.

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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 table Guide to disruption and homogenization methods for different samples 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.

Guide to disruption and homogenization methods for different samples
Starting material Disruption method  Homogenization method  Comments
 Cultured animal cells  Addition of lysis buffer  Rotor–stator homogenizer or syringe and needle for cytoplasmic RNA  If ≤1 x 105 cells are processed lysate can be homogenized by vortexing. No homogenization protocol.
 Animal tissue  Rotor–stator homogenizer  Rotor–stator homogenizer  Simultaneously disrupts and homogenizes.
 Animal tissue  Mortar and pestle  Syringe and needle  Rotor–stator homogenizer and bead mills usually gives higher yields than mortar and pestle.
 Animal tissue  Bead mill  Bead mill   
 Bacteria  Enzymatic (lysozyme) digestion followed by addition of lysis buffer  Vortex  If >5 x 108 cells are processed, further homogenization using a syringe and needle may increase yield.
 Bacteria  Bead mill  Bead mill  Bead milling simultaneously disrupts and homogenizes; bead milling cannot be replaced by vortexing.
 Yeast  Enzymatic (lysozyme/zymolase) digestion of cell wall followed by lysis of spheroplasts by addition of lysis buffer  Vortex     
 Yeast  Bead mill  Bead mill  Bead milling simultaneously disrupts and homogenizes; bead milling cannot be replaced by vortexing.
 Plants and filamentous fungi  Mortar and pestle  Syringe and needle  Mortar and pestle cannot be replaced by rotor–stator homogenizer.
 

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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 section, considerations for working with a number of different sources are discussed.

Plants

Isolation of RNA from plant material presents special challenges, and commonly used techniques often require adaptation before they can be used with plant samples. Several plant metabolites have chemical properties that are similar to nucleic acids, making them difficult to remove from RNA preparations. Co-purified metabolites (such as polysaccharides, polyphenolics, and flavones) and contaminants introduced by the purification procedure (such as salts or phenol) can inhibit enzymatic reactions or cause variations in UV spectrophotometric measurements and gel migration. Additional problems with RNA isolation from plant material include pipetting errors due to increased viscosity and RNA degradation during storage. 

RNA isolation is often improved by using plants grown under conditions that do not induce high levels of plant metabolites. Because of the great variation among plants, it is difficult to make general statements about growth conditions to use. However, as a general guideline, it is recommended to use healthy, young tissues when possible. RNA yields from young tissues are often higher than from old tissue because young tissue generally contains more cells than the same amount of older tissue. Young tissue of the same weight also contains fewer metabolites. In addition, many protocols for “home-made” RNA isolation methods recommend growing plants in darkness for 1 to 2 days before harvesting to prevent high-level accumulation of plant metabolites.

Heart, muscle, and skin tissue

RNA isolation from e.g., skeletal muscle, heart, and skin tissue can be difficult due to the abundance of contractile proteins, connective tissue, and collagen. In order to remove these proteins, which can interfere with RNA isolation, the sample needs to be treated with a protease or phenol-containing lysis reagents. However, the protease digest needs to be carried out under conditions that do not allow RNA degradation.

Bacteria

Bacterial mRNAs differ from eukaryotic mRNAs in a number of essential features. Prokaryotic mRNAs have no 5' cap and only rarely have poly-A tails. The absence of a poly-A tail means that mRNA isolation by hybrid capture is not possible. In addition, oligo-dT primers cannot be used to prime first-strand cDNA synthesis so random primers need to be used instead.

In addition, bacterial mRNAs are highly unstable, with an average half-life of about 3 minutes for fast-growing bacteria. Sometimes the bacterial mRNA begins to degrade while it is still being translated. This can be a big problem for researchers trying to isolate mRNA from bacteria. Since mRNAs are very rapidly turned over in bacteria, gene expression studies are even more difficult in prokaryotes than in eukaryotes. To accurately preserve gene expression patterns and to maximize the amount of fully intact mRNA isolated, samples need to be stabilized prior to sample harvesting and processing.

Blood

Blood samples are routinely collected for clinical analysis. RNA in blood samples can be preserved using an RNA stabilization reagent in the collection tube. RNA isolation from blood requires a method to provide high-quality RNA without contaminants or enzyme inhibitors. Blood contains a number of enzyme inhibitors that can interfere with downstream RNA analysis. In addition, common anticoagulants such as heparin and EDTA can interfere with downstream assays. It should be noted that anticoagulants do not provide RNA stabilization. 

Erythrocytes (red blood cells) of human blood do not contain nuclei and are therefore not important for RNA isolation since they do not synthesize and contain only very small amounts of RNA. The target of isolation from whole blood is leukocytes (white blood cells), which are nucleated and contain RNA. Leukocytes consist of 3 main cell types: lymphocytes, monocytes, and granulocytes.

Since healthy blood contains approximately 1000 times more erythrocytes than leukocytes, removing the erythrocytes simplifies RNA isolation. This can be accomplished by selective lysis of erythrocytes, which are more susceptible than leukocytes to hypotonic shock and burst rapidly in the presence of a hypotonic buffer.

A common alternative to erythrocyte lysis is Ficoll density-gradient centrifugation. In contrast to erythrocyte-lysis procedures, Ficoll density-gradient centrifugation only recovers mononuclear cells (lymphocytes and monocytes) and removes granulocytes. Mononuclear cells isolated by Ficoll density-gradient centrifugation can then be processed for RNA isolation as with other animal cells.

FFPE tissue samples

Formalin-fixed, paraffin-embedded (FFPE) tissue represents a valuable and extensive source of material for biomedical research. With more and more researchers turning toward molecular analysis of FFPE samples, it is becoming increasingly important to develop specific protocols that take into consideration the unique nature of these samples.

Due to fixation and embedding conditions, nucleic acids in FFPE samples are usually heavily fragmented and chemically modified by formaldehyde. Therefore, nucleic acids isolated from FFPE samples are often of a lower molecular weight than those obtained from fresh or frozen samples. The degree of fragmentation depends on the type and age of the sample and on the conditions for fixation, embedding, and storage of the sample. Although formaldehyde modification cannot be detected in standard quality control assays, such as gel electrophoresis or lab-on-a-chip analysis, it does strongly interfere with enzymatic analyses.

To minimize the effects of FFPE storage on RNA transcripts, the following tips should be borne in mind:

  • Remove and fix tissue as quickly as possible 
  • Use tissue samples no more than 5 mm thick and do not over-fix (max. 24 hours) 
  • Use high-quality reagents for paraffin embedding, without additives 
  • Avoid sample staining, if possible 
  • Store FFPE samples appropriately (RNA remains intact when stored at 4°C for up to 1 year, rather than at room temperature or higher) 
  • Use an appropriate deparaffinization step
  • RNA isolation should involve a crosslink-reversal step
RNA viruses

Some viruses have a single- or double-stranded RNA genome (see table Selected RNA viruses). Viral RNA is typically isolated from cell-free body fluids, where their titer can be very low. Virus particles may need to be concentrated by ultracentrifugation, ultrafiltration, or precipitation before RNA isolation. Addition of carrier RNA may also be necessary during RNA isolation when the expected yield of RNA is low.

A major problem with viral RNA is that it typically has a high degree of secondary structure. This can make downstream analysis especially difficult. Many reverse transcriptases have difficulty transcribing through complex RNA secondary structure. In addition, RNA viruses have a high mutation rate due to inaccurate copying when they replicate. Therefore it is often difficult to obtain a homogeneous population for analysis.

Cell-free RNA in plasma or serum (or other body fluids)

RNA, especially miRNA, associated with proteolipids (vesicles) or proteins can be found in plasma, serum, urine, other body fluids, and in cell culture supernatants. The concentration is much lower than that of cellular RNA, but approximately tenfold higher than cell-free DNA in human plasma.The RNA is relatively stable, with a half-life of about 2 days in human whole blood. Nonetheless, the RNA can be degraded by repeated freeze–thaw cycles. As with viral RNA in cell-free body fluids, addition of carrier RNA may be necessary during RNA isolation of this RNA.

Selected RNA viruses
Family  Selected viruses Genome
 Adenoviridae  Adenovirus  dsDNA
 Arenaviridae  Lassa virus  ssRNA
 Bornaviridae  Borna disease virus  ssRNA
 Bunyaviridae  Hantaan virus  ssRNA
 Caliciviridae  Hepatitis E virus, Norwalk virus  ssRNA
 Filoviridae  Ebola virus  ssRNA
 Flaviviridae  Hepatitis C and G viruses, Dengue virus  ssRNA
 Hepadnaviridae  Hepatitis B virus  ss/dsDNA
 Herpesviridae  Herpesviruses (HSV; CMV; EBV; HHV6, 7, 8)  dsDNA
 Papovaviridae  Human papillomavirus, JC virus  ssDNA
 Paramyxoviridae  Parainfluenza virus, respiratory syncytial virus, rubulavirus  ssRNA
 Parvoviridae  Parvovirus B19 (erythrovirus)  ssDNA
 Picornaviridae  Coxsackie virus, foot-and-mouth disease virus, hepatitis A virus, poliovirus, rhinovirus  ssRNA
 Reoviridae  Rotavirus  dsRNA
 Retroviridae  Human foamy virus, human immunodeficiency virus, human T-cell leukemia virus  ssRNA
 Rhabdoviridae  Rabies virus  ssRNA

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Storage of RNA

Purified RNA can be stored at –20°C or –70°C in RNase-free water. Under these conditions, no degradation of RNA is detectable after 1 year.

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Quantification of RNA 

See Spectrophotometric measurement of RNA concentration. As discussed in Determining RNA quality, the ratio between the absorbance values at 260 and 280 nm gives an indication of RNA purity.

Microvolume (or microdroplet) UV readers (e.g., Nanodrop) can also be used to determine RNA concentration.

When measuring RNA samples, be certain that cuvettes are RNase-free, especially if the RNA is to be recovered after spectrophotometry. This can be accomplished by washing cuvettes with 0.1 M NaOH, 1 mM EDTA followed by washing with RNase-free water (see Solutions). Use the buffer in which the RNA is diluted to zero the spectrophotometer.

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Spectrophotometric measurement of RNA concentration

The concentration of RNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer using UV-permeable plastic cuvettes. To ensure significance, readings should be between 0.15 and 1.0. An absorbance of 1 unit at 260 nm corresponds to 44 µg of RNA per ml (A260 = 1 → 44 µg/ml; based on a standard 1 cm path length). This relation is valid only for measurements made at neutral pH. Therefore, if it is necessary to dilute the RNA sample, this should be done in a low-salt buffer with neutral pH (e.g., 10 mM Tris·Cl, pH 7.0).

When measuring RNA samples, be certain that cuvettes are RNase-free, especially if the RNA is to be recovered after spectrophotometry. This can be accomplished by washing cuvettes with 0.1 M NaOH, 1 mM EDTA, followed by washing with RNase-free water (see Solutions). Use the buffer in which the RNA is diluted to zero the spectrophotometer. An example of the calculation involved in RNA quantification is shown below:

Example of RNA quantitation
Volume of RNA sample = 100 µl
Dilution = 10 µl RNA sample + 490 µl of 10 mM Tris·Cl, pH 7.0 (1/50 dilution)
Measure absorbance of diluted sample in a 1 ml cuvette (RNase-free)
A260 = 0.2

RNA concentration
= 44 µg/ml x A260 x dilution factor
= 44 µg/ml x 0.2 x 50
= 440 µg/ml

Total amount of RNA
= concentration x volume of sample in ml
= 440 µg/ml x 0.1 ml
= 44 µg RNA

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Determining RNA quality

RNA purity

The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV, such as protein. However, the A260/A280 ratio is influenced considerably by pH. Since water is not buffered, the pH and the resulting A260/A280 ratio can vary greatly. Lower pH results in a lower A260/A280 ratio and reduced sensitivity to protein contamination (3).

For accurate ratios, we recommend measuring absorbance in a low-salt buffer with slightly alkaline pH (e.g., 10 mM Tris·Cl, pH 7.5). Pure RNA has an A260/A280 ratio of 1.9–2.1 in 10 mM Tris·Cl, pH 7.5.

Note: Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some spectrophotometers.

Always be sure to calibrate the spectrophotometer with the same solution. For determination of RNA concentration, however, we still recommend dilution of the sample in a buffer with neutral pH since the relationship between absorbance and concentration (A260 reading of 1 = 44 µg/ml RNA) is based on an extinction coefficient calculated at neutral pH (see Quantification of RNA).

RNA integrity

The integrity and size distribution of total RNA can be checked by denaturing agarose gel electrophoresis (see RNA analysis: Analytical gels), ethidium bromide staining or using a commercially available system (for example, the QIAxcel system or Agilent 2100 Bioanalyzer). The respective ribosomal bands (see table Size of ribosomal RNAs from various sources) should appear as sharp bands on the stained gel. 28S ribosomal RNA bands should be present with an intensity approximately twice that of the 18S rRNA band (see figure Analysis of total RNA). If the ribosomal bands in a given lane are not sharp, but appear as a smear of smaller sized RNAs, it is likely that the RNA sample suffered major degradation during preparation.

The Agilent 2100 Bioanalyzer also provides an RNA Integrity Number (RIN) as a useful measure of RNA integrity. Ideally, the RIN should be close to 10, but in many cases (particularly with tissue samples), RNA quality is mainly influenced by how well the original sample was preserved.  

Size of ribosomal RNAs from various sources
Source  rRNA Size (kb)
 E. coli  16S
 23S
 1.5
 2.9
 S. cerevisiae  18S
 26S
 2.0
 3.8
 Mouse  18S
 28S
 1.9
 4.7
 Human  18S
 28S
 1.9
 5.0
 

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RNA analysis: Analytical gels 

Principle of denaturing gel analysis

Formaldehyde agarose gels allow separation and identification of RNA based on charge migration. Unlike DNA, RNA has a high degree of secondary structure, making it necessary to use a denaturing gel. Formaldehyde in the gel disrupts secondary RNA structure so that RNA molecules can be separated by their charge migration. 

In an electric field, nucleic acid molecules migrate towards the anode due to negatively charged phosphates along the backbone. The migration of denatured RNA molecules is determined by their size; however, the relationship between the fragment size and rate of migration is nonlinear, since larger fragments have a greater frictional drag and are less efficient at migrating through the gel.  

Agarose gel analysis is the most commonly used method for analyzing RNA species, which generally correspond in size to the resolution range of an agarose gel. Small RNA fragments, such as tRNAs or 5S rRNAs, can be analyzed by polyacrylamide gel electrophoresis. Detailed information on all types of analytical gels can be found in molecular biology manuals (2, 4). This section describes formaldehyde agarose gel electrophoresis.  

Preparing formaldehyde agarose gels for RNA analysis

The following protocol for formaldehyde agarose (FA) gel electrophoresis gives enhanced sensitivity for gel and subsequent analysis (e.g., northern blotting). A key feature of this protocol is the concentrated RNA loading buffer that allows a larger volume of RNA sample to be loaded onto the gel than provided for in conventional protocols.

Agarose

The concentration of agarose used for the gel determines the size range of RNA fragments that can be resolved. For most RNA species of interest, a concentration of 1.0–1.2% (w/v) agarose will give best results. For resolution of large mRNA species, it may be helpful to reduce the agarose concentration. For analysis of smaller mRNAs, the agarose concentration can be raised to 2%. With small RNA species, such as tRNAs or 5S rRNAs, polyacrylamide gel electrophoresis is recommended.

Use ultrapure agarose since impurities such as polysaccharides, salts, and proteins can affect the migration of RNA.

Tip: Plastics used for some electrophoresis tanks are not resistant to ethanol. Take proper care and check the supplier’s instructions.

Pouring the gel
  1. Prepare enough 10x FA gel buffer (see table FA gel buffer) to pour the gel and to make enough FA gel running buffer (see table FA gel running buffer) to fill the electrophoresis tank.
  2. Mix an appropriate amount of agarose, 10x FA gel buffer, and RNase-free water in a flask or bottle.
    To prepare FA gel of size 10 x 14 x 0.7 cm, mix:
    1.0–1.2 g agarose
    10 ml 10x FA gel buffer
    Add RNase-free water to 100 ml
    The vessel should be no more than half full. Cover the vessel to minimize evaporation.
    Tip: If smaller or larger gels are needed, adjust the quantities of components proportionately.
  3. Heat the mixture in a microwave or boiling water bath, swirling the vessel occasionally until the agarose is dissolved.
    Tip: Ensure that the lid of the flask is loose to avoid buildup of pressure. Be careful not to let the agarose solution boil over as it becomes super-heated.
    Tip: If the volume of liquid reduces considerably during heating due to evaporation, make up to the original volume with RNase-free distilled water. This will ensure that the agarose concentration is correct.
  4. Cool the agarose to 65–70°C in a water bath. Stir or swirl occasionally to prevent uneven cooling.
  5. After cooling, add 1.8 ml of 37% (12.3 M) formaldehyde and 1 µl of a 10 mg/ml ethidium bromide stock solution.
    Tip: Formaldehyde is toxic. Use a fume hood to avoid inhalation. Wear gloves and take appropriate safety precautions when handling.
    Tip: Make sure that the solution has cooled sufficiently before adding formaldehyde and ethidium bromide. Formaldehyde is volatile and may evaporate if added to a solution that is too hot.
    Tip: Ethidium bromide in the gel allows visualization of the RNA with UV light. Ethidium bromide is toxic and a powerful mutagen. Wear gloves and take appropriate safety precautions when handling. Use of nitrile gloves is recommended as latex gloves may not provide full protection. After use, ethidium bromide solutions should be decontaminated as described in commonly used manuals.
    Tip: Stock solutions of ethidium bromide (generally 10 mg/ml in water) should be stored at 2–8°C in a dark bottle or a bottle wrapped in aluminum foil.
  6. Pour the agarose solution onto the gel tray in a fume hood to a thickness of 3–5 mm. Insert the comb either immediately before or immediately after pouring. Let the gel set for at least 30 min.
    Tip: Ensure that there is enough space between the bottom of the comb and the gel tray (0.5–1.0 mm) to allow proper well formation and avoid sample leakage.
    Tip: Make sure that there are no air bubbles in the gel or trapped between the wells. Air bubbles can be carefully removed with a Pasteur pipet before the gel sets.
    Tip: Thicker gels can be used to increase the amount of sample volume that can be loaded. Thinner gels generally transfer better in northern blotting, but smaller sample volumes can be used.
    Tip: The thickness of the comb affects the sharpness of bands in the gel. A thinner comb gives sharper bands, but less sample can be loaded per well.
  7. Leaving the comb in the gel, place the gel in the electrophoresis tank. Fill the tank with 1x FA gel running buffer.
    Tip: Add enough buffer to cover the gel with approximately 1 mm of liquid above the surface of the gel. If too much buffer is used, the electric current will flow through the buffer instead of the gel.
  8. Carefully remove the comb from the gel. Prior to running, let the gel equilibrate in 1x FA gel running buffer for at least 30 min.

FA gel buffer*
Composition of 10x working solution  Component Amount per liter
 200 mM MOPS  MOPS free acid  41.9 g
 50 mM sodium acetate  Sodium acetate·H2O  6.8 g
 10 mM EDTA  0.5 M EDTA, pH 8.0  20 ml
Adjust to pH 7.0 using NaOH.
* FA gel buffer turns yellow during autoclaving; this has no effect on gel electrophoresis.
Alternatively, 4.1g anhydrous sodium acetate,
 

FA gel running buffer
Composition of 1x working solution  Component Amount per liter
 1x FA gel buffer  10x FA gel buffer  100 ml
 0.25 M formaldehyde*  37% (12.3 M) formaldehyde  20 ml
 –  RNase-free water  880 ml
* Toxic and/or mutagenic. Take appropriate safety measures.
See “Preparation of RNase-free solutions”.
 

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Running and analyzing formaldehyde gels for RNA analysis

RNA loading buffer

RNA loading buffer (see table RNA loading buffer) must be added to samples before loading them on a gel. The loading buffer serves three main purposes:

  • To denature the RNA sample prior to loading. 
  • To increase the density of the samples to ensure that they sink into the wells on loading. 
  • To add color to the samples through the use of dyes, facilitating loading and visualization on the gel while running.

A key feature of the concentrated RNA loading buffer is that it allows a larger volume of RNA sample to be loaded onto the gel than conventional protocols allow.

RNA loading buffer
Composition of 5x working solution  Component Amount per 10 ml
 0.25% bromophenol blue  Bromophenol blue  25 mg*
 4 mM EDTA  0.5 M EDTA, pH 8.0  80 µl
 0.9 M formaldehyde  37% (12.3 M) formaldehyde  750 µl
 20% glycerol  Glycerol  2 ml
 30.1% formamide  Formamide  3.084 ml
 4x FA gel buffer  10x FA gel buffer  4 ml
Stable for ~3 months at 2–8°C.
* Alternatively, use 16 µl of a saturated aqueous bromophenol blue solution instead of 25 mg powder. To make this solution, add solid bromophenol blue to distilled water, mix, and continue to add more bromophenol blue until no more will dissolve. Centrifuge to pellet the undissolved powder, and carefully pipet the saturated supernatant.
Toxic and/or mutagenic. Take appropriate safety measures.
 

Electrophoresis buffers

RNA gels are run at a lower pH than DNA gels since RNA has a lower pKa than DNA. Furthermore, unlike DNA, RNA is susceptible to alkali cleavage at high pH. RNA gels should therefore be run at neutral pH. MOPS (3-[N-morpholino]propanesulfonic acid) is the most commonly used buffer for RNA gels due to its high buffering capacity at pH 7.0. Formaldehyde is included in the running buffer to keep the RNA denatured. Formaldehyde is also added to the agarose gel.

Note: Electrophoresis tanks should be cleaned with detergent solution (e.g., 0.5% SDS), thoroughly rinsed with RNase-free water, and then rinsed with ethanol and allowed to dry. However, plastics used for some electrophoresis tanks are not resistant to ethanol. Take proper care and check the supplier’s instructions.

Sample preparation for electrophoresis
  1. Add 1 volume of 5x RNA loading buffer to 4 volumes of RNA sample (e.g., 5 µl loading buffer and 20 µl RNA) and mix.
    Samples should always be mixed with RNA loading buffer prior to loading on a gel.
    Tip: Do not use sample volumes close to the capacity of the wells as samples may spill over into adjacent wells during loading.
    Tip: Be sure that all samples have the same buffer composition. High salt concentrations will retard the migration of RNA molecules.
    Tip: Ensure that no ethanol is present in the samples, for example, carried over from purification procedures.
    Tip: Ethanol may cause samples to float out of the wells on loading.
  2. To denature RNA, incubate for 3–5 min at 65°C. Chill on ice.
Electrophoresis
  1. Apply denatured samples to the wells of the gel. The gel should be submerged in electrophoresis buffer in the electrophoresis tank prior to loading.
    Tip: Prior to sample loading, remove air bubbles from the wells by rinsing them with electrophoresis buffer.
    Tip: Make sure that the entire gel is submerged in the FA gel running buffer.
    Tip: To load samples, insert the pipet tip deep into the well and expel the liquid slowly. Take care not to break the agarose with the pipet tip.
    Tip: Once samples are loaded, do not move the gel tray/tank as this may cause samples to float out of the wells.
    Tip: Be sure to include at least one lane of appropriate molecular-weight markers.
  2. Connect the electrodes of the electrophoresis apparatus so that the RNA will migrate towards the anode or positive lead (usually red).
    Tip: The electrophoresis apparatus should always be covered to protect against electric shock.
    Tip: Run the gel in a fume hood to avoid exposure to formaldehyde fumes from the gel and running buffer.
  3. Turn on the power supply, and run the gel at 5–7 V/cm until the bromophenol blue dye has migrated approximately 2/3 of the way through the gel.
    Tip: Avoid use of high voltages, which can cause trailing and smearing of RNA bands.
    Tip: Monitor the temperature of the buffer periodically during the run. High temperature can cause partial melting of the gel and distortion of the bands. If the buffer becomes significantly heated, reduce the voltage.
    Tip: For very long runs (e.g., overnight runs), use a pump to recycle the buffer.
Visual analysis of the gel

Ethidium bromide in the gel allows visualization of the RNA with UV light. After use, ethidium bromide solutions should be decontaminated as described in commonly used manuals (2, 4).

Stock solutions of ethidium bromide (generally 10 mg/ml in water) should be stored at 2–8°C in a dark bottle or a bottle wrapped in aluminum foil.

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.

Visualization

Ethidium bromide–RNA complexes display increased fluorescence compared to the uncomplexed dye in solution. This means that illumination of a stained gel under UV light (254–366 nm) allows bands of RNA to be visualized against a background of unbound dye. The gel image can be recorded by taking a Polaroid photograph or using a gel documentation system.  

Tip: UV light can damage the eyes and skin. Always wear suitable eye and face protection when working with a UV light source. 

Tip: UV light damages RNA. If RNA fragments are to be extracted from the gel, use a lower intensity UV source if possible, and minimize exposure of RNA to the UV light. 

Analysis of total RNA

The integrity and size distribution of total RNA can be checked by observing the stained RNA. The respective ribosomal bands should appear as sharp bands on the stained gel (see table Size of ribosomal RNAs from various sources and figure Analysis of total RNA). If the ribosomal bands in a given lane are not sharp, but appear as a smear towards smaller sized RNAs, it is likely that the RNA sample suffered major degradation during preparation. The 28S ribosomal RNA band should be present at approximately twice the intensity of the 18S rRNA band. Since the 28S rRNA is more labile than the 18S rRNA, equal intensities of the 2 bands generally indicates that some degradation has occurred.

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RNA analysis: Northern blotting

After separating RNA molecules based on charge migration in a denaturing gel, RNA molecules in the gel are transferred to a nylon or nitrocellulose membrane by capillary transfer. The RNA of interest can then be identified by hybridization to radioactive or chemoluminescent probes and visualized by autoradiography or photography.

Since DNA blotting is commonly referred to as “Southern blotting”, after its inventor E.M. Southern, the term “northern blotting” was coined for this analogous RNA-blotting process.

The northern blotting procedure

The following is one protocol for northern blotting. This procedure is intended for use with a standard formaldehyde agarose gel, prepared and run as described in RNA analysis: Analytical gels.

Blotting membrane

Northern blotting is generally carried out by immobilization of the RNA on nylon or nitrocellulose membranes. Positively charged nylon membranes are generally recommended over nitrocellulose because of their greater strength and ease of handling.

Tip: Always wear gloves while working with blotting membranes. Handle membranes carefully by the edges or using clean blunt-ended forceps.

Transfer buffer

Formaldehyde agarose RNA gels are generally blotted using a high-salt buffer such as 20x SSC (see table Northern transfer solution). Prepare 1.2 liters of 20x SSC. For larger RNA gels (>100 ml volume), use 2.4 liters of 20x SSC.

Tip: Save 200 ml of the 20x SSC solution, and dilute it twofold to make 10x SSC for soaking the gel and washing the blot after transfer.

Northern transfer solution 
 Composition of working solution, 20x SSC Component Amount per liter
 3 M NaCl  NaCl  175.3 g
 0.3 M sodium citrate  Sodium citrate·2H2O  88.2 g
Adjust to pH 7.0 with NaOH.
 

Equipment required

  • Whatman 3MM filter paper 
  • Paper towels, a stack of approximately 15–20 cm 
  • Plastic wrap 
  • Two glass or Plexiglas plates 
  • Buffer tray (e.g., glass casserole dish) capable of holding 1–2 liters of buffer 
  • Flat weight, approximately 1 kg 
  • RNase-free water (200 ml) 
  • 0.05 M NaOH (200 ml)
Presoaking filter paper and blotting membrane
  1. Cut one sheet of nylon membrane and two sheets of Whatman 3MM paper about 1 mm larger than the gel on each edge.
  2. Cut two lengths of Whatman paper wider than the gel, long enough to fit under the gel and reach to the bottom of the dish on either side (see figure Northern blot setup).
  3. Wet the nylon membrane in water. Then soak the Whatman paper and nylon membrane in 20x SSC for 1–2 min.
Capillary transfer
  1. Fill the buffer tray with 1 liter 20x SSC. Place a glass or Plexiglas plate across the tray or on top of a support (see figure Northern blot setup).
  2. Place the two lengths of presoaked filter paper over the glass or Plexiglas plate so that the ends contact the bottom of the tray. Remove any air bubbles between the sheets of filter paper and the plate by rolling a pipet several times back and forth over the surface.
  3. Immediately after gel electrophoresis, soak the gel for 10 min, with gentle shaking, in 200 ml RNase-free water and then for 15 min in 200 ml 0.05 M NaOH. Finally, soak the gel for 10 min in 200 ml 10x SSC to neutralize the NaOH.
    Tip: Dilute 20x SSC twofold to make 10x SSC.
    Tip: The gel contains formaldehyde to denature the RNA. Formaldehyde is toxic. Use a fume hood to avoid inhalation. Wear gloves and take appropriate safety precautions.
  4. Position the gel upside-down on the filter paper covering the plate.
  5. Place a sheet of plastic wrap over the gel. Use a sheet large enough to cover the surface of the filter paper on the glass or Plexiglas plate. Using a clean scalpel or razor blade, carefully cut the plastic wrap around the gel. Remove the piece over the gel so that the remaining plastic wrap surrounds the gel. This ensures that the transfer buffer moves only through the gel and not around it.
  6. Place the presoaked nylon membrane on top of the gel so that it covers the entire surface. Do not move the nylon membrane once it has been placed on the gel. Remove any air bubbles between the membrane and the gel by gently rolling a pipet several times back and forth over the surface.
  7. Place the two presoaked sheets of Whatman 3MM paper on top of the nylon membrane. Again, remove any air bubbles by gently rolling a pipet several times back and forth over the surface.
  8. Place a 15–20 cm stack of dry paper towels on top of the filter paper.
    Tip: Make sure that the plastic wrap around the gel prevents contact of the paper towels with the transfer buffer and the wet filter paper under the gel. Ensure that the towels do not droop over since they can cause liquid to flow around the gel instead of through it.
  9. Place a second glass or Plexiglas plate on top of the paper towels. Place the 1 kg weight on top of the plate.
  10. Let the transfer proceed for 12–18 h.
    Tip: Remove the wet paper towels and replace them with dry ones at least once during the transfer. If necessary, add more transfer buffer to the buffer tray.
    Tip: The gel contains formaldehyde which will diffuse out of the gel during transfer. Formaldehyde is toxic. Perform the transfer in a fume hood to avoid inhalation. Wear gloves and take appropriate safety precautions when handling.
Fixing the RNA to the blot
  1. After the transfer is complete, remove the weight, paper towels, and the two sheets of filter paper. Turn over the gel and the nylon membrane together, and lay them, gel-side up, on a dry sheet of filter paper. Mark the positions of the gel lanes on the membrane using a ball-point pen or a soft-lead pencil. Peel the gel from the membrane and discard it.
    Tip: Make sure to mark the gel lanes before removing the gel from the nylon membrane! Without this marking, you won’t be able to tell which lane is which.
    Tip: Most of the formaldehyde in the gel transfers into the paper towels and the upper sheets of filter paper.
    Tip: Dispose of them according to your institution’s waste-disposal guidelines.
  2. Wash the nylon membrane for 1 min in 100–200 ml 10x SSC.
    Tip: Dilute 20x SSC twofold to make 10x SSC. This wash step is critical to remove any agarose that adheres to the blot.
  3. Fix the RNA to the blot by baking (step 4) or UV-crosslinking (step 5).
    Tip: UV-crosslinking generally gives better results and enhanced sensitivity compared to baking. However, proper crosslinking requires prior optimization of the system.
  4. To fix the RNA by baking, first let the blot air-dry on a dry sheet of filter paper, then place between two sheets of filter paper. Bake for 30 min to 2 h at 80°C in a vacuum oven.
  5. To fix the RNA by UV-crosslinking, take the damp blot and expose the side with the RNA to UV irradiation (e.g., with a UV transilluminator) for a determined length of time.
    Tip: To determine the proper conditions for UV irradiation, the system must first be empirically tested and optimized. To do this, take an RNA blot with several lanes containing identical RNA samples. Cut the blot into separate strips for each lane, and irradiate each for different times, varying from 0.5 to 5 min. After hybridization, determine which time gives the optimal signal intensity. Be sure to use the same conditions (UV wavelength, distance from UV source) for each experiment. In addition, the system should be routinely calibrated to determine that the intensity of the UV irradiation remains unchanged.
    Tip: UV light can damage the eyes and skin. Always wear suitable eye and face protection.
  6. If the blot is not to be used immediately, store it at 4°C, wrapped in plastic wrap.

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Capillary electrophoresis

The integrity of RNA molecules is of paramount importance for gene expression analysis experiments and using intact RNA is a key step in obtaining meaningful experimental data.

Background

Historically, RNA integrity has been evaluated using the ratio of 28S:18S rRNA using agarose gel electrophoresis stained with ethidium bromide. This produces a defined banding pattern (4). Typically, a gel image will show two bands comprising the 28S and 18S rRNA species and other bands where smaller RNA species are located. RNA is considered of high quality when the ratio of 28S:18S bands is ≥2.0.

However, this method is inconsistent; it is a subjective measure which relies on the human eye for interpretation. Therefore, it cannot be standardized between, or even within, laboratories.

The introduction of microcapillary electrophoretic RNA separation has provided the basis for an automated approach to determine the integrity of RNA samples in a more standardized, less ambiguous way.

Capillary electrophoresis enables rapid size-based separation of nucleic acids. Unlike traditional agarose gel electrophoresis, separation is performed in a capillary of a ready-to-use gel cartridge. The samples are automatically loaded into an individual capillary and voltage is applied. The negatively charged nucleic acid molecules migrate through the capillary to the positively charged terminus. As with agarose gel electrophoresis, low-molecular-weight molecules migrate faster than high-molecular-weight molecules. As the molecules migrate though the capillary, they pass a detector which detects and measures the fluorescent signal. A photomultiplier converts the fluorescent signal into electronic data, which are then transferred to a computer for further processing.

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RNA integrity parameters

RNA integrity can be measured in a number of ways, including the RNA Quality Indicator (RQI), RNA Quality Score (RQS) and RNA Integrity Number (RIN). RIN is currently the commonest method.

RNA Integrity Number (RIN)

The RNA integrity number (RIN) is a software algorithm designed to help scientists estimate the integrity of total RNA samples. The software automatically assigns an integrity number to a eukaryotic total RNA sample.

Using this tool, sample integrity is no longer determined by the ratio of the ribosomal bands, but by the entire electrophoretic trace of the RNA sample. This includes the presence or absence of degradation products. In this way, interpretation of an electropherogram is facilitated, comparison of samples is enabled, and repeatability of experiments is ensured. The assigned RIN is independent of sample concentration, instrument, and analyst; therefore a genuine standard for RNA physical integrity.

RIN provides:

  • A numerical assessment of the integrity of RNA.
  • A direct RNA sample comparison (e.g., between laboratories).
  • Experimental repeatability (e.g. if RIN shows a value suitable for microarray experiments, then a RIN of the same value can always be used for similar experiments given that the same tissue and extraction method are used).
Using RIN in practice

Firstly, the RIN values must be validated. This can be done by correlating RIN numbers with a specific downstream experiment, such as microarray analysis or RT-PCR. This correlation step can be used to establish a RIN threshold value between successful downstream experiments and failed experiments. It can be performed on sets of existing bioanalyzer data, if available. After the threshold value has been established, this value can be used in the standard RNA QC procedure. All samples with a RIN higher than the threshold value pass the QC test, while samples below the threshold value are discarded. The correlation step of validating the RIN has to be repeated if a significant experimental parameter is changed (including, for example, different organism investigated, different type of microarray used, using different probe sets, etc.).

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Removal of genomic DNA contamination from RNA

No currently available purification method can guarantee that RNA is completely free of DNA, even when it is not visible on an agarose gel. For analysis of very low abundance targets, any interference by residual DNA contamination can be detected by performing real-time RT-PCR control experiments in which no reverse transcriptase is added prior to the PCR step. To prevent any interference by DNA in real-time RT-PCR applications, we recommend designing primers that anneal at intron splice junctions so that genomic DNA will not be amplified. DNA can also be removed during cDNA synthesis with some commercial kits. For other sensitive applications, DNase digestion of the purified RNA with RNase-free DNase is recommended.

References

  1. Alberts, B., et al. (2002) Molecular Biology of the Cell. 4th ed. New York: Garland Publishing, Inc.
  2. Ausubel, F.M. et al. (1991) Current Protocols in Molecular Biology, New York: John Wiley and Sons.
  3. 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.
  4. 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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