- Email: [email protected]
Basic molecular biology
PAEDIATRIC RESPIRATORY REVIEWS (2005) 6, 199–208
SERIES: BASIC SCIENCE RESEARCH IN RELATION TO THE LUNG
Basic molecular biology Albert P. Senft and Ann Marie LeVine* Divisions of Neonatology and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave.,Cincinnati, OH 45229, USA KEYWORDS Nucleic acids; Proteins; PCR; Immunohistochemistry
Summary Rapid advances in molecular biology over the past 20 years have and will continue to impact on the practice of medicine. Advances in molecular biology are having an immense impact in determining the underlying aetiology of lung disease and its treatment. In this review, basic molecular biology techniques will be discussed with examples of how these techniques are used in clinical practice. ß 2005 Elsevier Ltd. All rights reserved.
INTRODUCTION Molecular biology is a broad area of study aimed at the common goal of understanding the mechanisms of basic cellular function. This review has been written as an introduction to the basic molecular biology techniques used to study DNA, RNA and proteins. The techniques utilised in basic and clinical research as well as the strengths and weaknesses of each technique are described. In addition, a bibliographical list of reference materials for an indepth description of molecular biology techniques has been included.
NUCLEIC ACIDS DNA encodes the molecular template for all molecules necessary for cell function and viability. The nucleotide is the most basic structural unit of DNA and is comprised of deoxyribose sugar, a phosphate group and a purine (adenine and guanine) or pyrimidine (thymine and cytosine) nitrogenous base. DNA is a double helical molecule with two sugar–phosphate strands serving as the backbone of the molecule with the nitrogenous bases projecting toward
* Corresponding author. Tel.: +1 513 636 2995; Fax: +1 513 636 7868. E-mail address: [email protected] (A.M. LeVine). 1526-0542/$ – see front matter ß 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.prrv.2005.06.006
the centre. The two strands are held together by hydrogen bonding, a non-covalent interaction, between the purine and pyrimidine bases. The interaction between the bases occurs with high fidelity (adenine always pairs with thymine and guanine with cytosine). Importantly, the two strands of the DNA molecule can be disassociated and then reassociated with the addition or removal of energy to break the forces of hydrogen bonding. Together these two physical features make DNA a dynamic molecule that allows for its replication and for transcription of encoded genes. The gene is the smallest functional unit of DNA and encodes for proteins. In general, genes are made up of a promoter region and a coding region (Fig. 1). The promoter region of the gene contains short, conserved, defined nucleotide sequences that positively or negatively regulate gene transcription. Gene transcription is a tightly regulated process that is cell-type specific and greatly influenced by the physiological stress placed upon the cell. Gene transcription produces a ribonucleic acid (RNA) template from which a protein is produced. RNA, like DNA, contains a sugar–phosphate backbone and nitrogenous bases. The major differences are that the sugar is ribose, the base uracil is incorporated in place of thymine and the molecule is single-stranded. Total cellular RNA is a mixture of three types: transfer (tRNA), ribosomal (rRNA) and messenger (mRNA), which is the
200
A. P. SENFT AND A. M. LEVINE
Figure 1
A hypothetical gene, primary transcript and final mRNA.
template for protein production. Since mRNA encodes for proteins, this will be the focus of the remaining discussion of RNA. Initial transcription results in the formation of a primary transcript that includes introns, exons and the 30 untranslated region (30 UTR) (Fig. 1). Exons are segments that encode the amino acid sequence of the protein. Introns are intervening sequences that do not encode a protein but guide splicing of the mature mRNA and allow alternative combinations of exons to produce variations of similar proteins. Excision of introns and splicing of exons must occur before mRNA protein translation can occur.
Methods for the Analysis of Nucleic Acids Polymerase Chain Reaction Polymerase chain reaction (PCR) is used to amplify specific DNA sequences from limited biological samples and has revolutionised biomedical research. The technique is based on the following: DNA can be denatured from a doublestranded molecule to a single-stranded molecule by heat. When the single-stranded DNA molecules are cooled they anneal to a double-strand, the process of two single-
stranded DNA molecules annealing occurs with high fidelity (i.e. A always pairs with T and C with G). The polymerase chain reaction is illustrated in Fig. 2. Sequentially, the template DNA is denatured by heating. Specific primers designed for the sequence of interest anneal on the corresponding DNA sequence of the template DNA when the reaction is cooled. DNA polymerase extends the DNA from 50 to 30 building a mirror image of the template strand. At the end of one cycle, two exact copies of template DNA have been formed. Using this technique, DNA can be amplified exponentially. This allows minute amounts of DNA from biological samples to be assayed, making the technique extremely powerful for basic science and clinical research. PCR is a common approach used for the diagnosis of clinical diseases. For example, PCR can be used for the detection of herpes simplex virus in cerebral spinal fluid1 and enterovirus in myocardial biopsy samples.2
Northern blot analysis Northern blot analysis is a sensitive technique for detecting mRNA expression levels. RNA is separated by agarose gel electrophoresis. Since, RNA has a negative charge it
BASIC MOLECULAR BIOLOGY
Figure 2
201
The steps involved in a polymerase chain reaction (PCR).
migrates to the cathode when a current is applied to the gel. Similar to other forms of electrophoresis, mobility of the molecule in the gel is directly proportional to its size. After separation by electrophoresis the RNA is transferred to a nitrocellulose membrane and cross-linked to the membrane by ultra-violet (UV)-irradiation. To determine mRNA levels for a specific gene, a short DNA probe is designed for the specific mRNA of interest. The probe is radiolabelled for detection and hybridised with a nitrocellulose membrane binding the mRNA molecules with a similar sequence. The results of hybridisation are detected by
autoradiography and reveal the number, size and abundance of transcripts (Fig. 3). While this technique is time consuming and requires a significant amount of RNA for analysis, it is still commonly used because it is extremely quantitative and therefore provides an accurate assessment of mRNA levels.
Reverse Transcriptase–Polymerase Chain Reaction Reverse transcriptase–polymerase chain reaction (RT-PCR) is used to assess the level of mRNA expression. As its
202
A. P. SENFT AND A. M. LEVINE
visualised using UV light (Fig. 5A). An important advantage of RT-PCR is that only a small amount of biological sample is necessary to determine the mRNA level. The limitation of this method for determining gene expression is that RT-PCR combined with visualisation using ethidium bromide is only semi-quantitative due to the fact that the PCR reaction is linear only within a range of cycles. While RT-PCR is not well suited for the quantitative comparison of mRNA expression, the technique is useful in screening tissues for the expression of a gene. For example, RT-PCR based assays are used in the clinical setting for the detection of RNA viruses, including respiratory syncytial virus and influenza A virus.3
Real-Time PCR
Figure 3
The steps involved in a Northern blot analysis.
name indicates, a portion of the assay relies on the amplification of a DNA template by PCR. Before PCR amplification, the mRNA is converted into a complimentary DNA (cDNA) molecule. As illustrated in Fig. 4, the enzyme reverse transcriptase is used to make a DNA copy of the mRNA. mRNA is selectively copied by using a primer that binds to the poly A tail of mRNA (not present in the more abundant rRNA and tRNA). Once the cDNA is made, the RNA is degraded by RNAse H. DNA polymerase and T4 DNA polymerase are then used to build the complementary strand. The result is a collection of DNA molecules that represents all the specific mRNA contained in the cell. Similar to the PCR of DNA, primers are designed for a specific gene and, using these primers, the product is exponentially amplified by PCR. The resulting PCR product is subjected to agarose gel electrophoresis to separate the PCR products by size. Ethidium bromide intercalates into the DNA and can be
The technology of real-time PCR developed out of the necessity for a quantitative technique to determine gene expression levels in small samples. Measurement of mRNA for a particular gene by real-time PCR is not fundamentally different from reverse-transcriptase PCR. Both techniques require mRNA initially to be reverse transcribed to a cDNA molecule. The cDNA is then subjected to PCR. The difference lies in the fact that the instrument where the PCR reaction is carried out is both a thermocycler and a detection device. The reaction mix contains a fluorescent probe that intercalates into the DNA during synthesis. At the end of each PCR cycle, the amount of fluorescence is assessed. Based on an algorithm, a threshold level of fluorescence must be reached for an mRNA to be considered expressed and this is expressed as a cycle number (Fig. 5B). mRNA expression is therefore directly related to cycle number; the higher the relative expression the lower the cycle number where the curve crossed the threshold. For example, in Fig. 5B, gene A has a higher expression than gene B because the cycle number, where it crossed the threshold, is lower.
PROTEINS Proteins are the molecules that perform all cellular functions. They are translated from the mRNA by ribosomes. Each amino acid is encoded by at least one triplet nucleotide sequence. Protein translation begins with the codon AUG, which encodes for methionine and is terminated with the stop codons UAA, UGA or UAG. Following translation, proteins are modified (e.g. by glycosylation) and sorted to their appropriate location (e.g. soluble or membranous) by a highly ordered process. With the advent of high-throughput techniques (e.g. microarrays that will be discussed in a future review), examining changes in gene expression under various experimental and pathophysiological conditions has provided a wealth of information and a basis for exploring the
BASIC MOLECULAR BIOLOGY
Figure 4
203
The generation of cDNA from mRNA by the reverse transcriptase reaction.
underlying mechanisms of disease processes. However, changes in gene expression only represent potential changes in protein levels and/or cellular function. Basic approaches to protein analysis are therefore important to understand whether changes in gene expression correlate to changes in protein expression and altered cellular function.
Methods for the Analysis of Proteins Sodium dodecyl sulphate polyacrylamide gel electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique that is used to separate
proteins based on their molecular weight. The detergent SDS has a negative charge and when added to the sample it binds hydrophobic amino acid residues. Proteins bind proportionally to the same amount of SDS. Therefore, the charge to mass of all proteins is equivalent and all SDS– protein complexes are negative. Protein samples are prepared either under reducing conditions where b-mercaptoethanol is added and the sample is boiled, or under nonreducing conditions where b-mercaptoethanol and heat are omitted. The specific sample preparation conditions are determined by experimental necessity. Once the samples have been prepared for electrophoresis, the sample is loaded on the gel and an electric field is applied. The rate of migration of the protein is inversely proportional to the logarithm of its molecular weight, meaning larger proteins
204
A. P. SENFT AND A. M. LEVINE
the primary antibody, the membrane is incubated with an antibody that is conjugated to horseradish peroxidase and is capable of recognising the primary antibody. For example if the antibody against protein X was raised in a rabbit, the second antibody used would be an anti-rabbit horseradish peroxidase conjugated antibody. The highly specific antibody to protein binding is detected using a peroxidase-dependent luminescence generating system followed by exposure to X-ray film. The bands that appear reflect the size and relative amount of the studied protein (Fig. 6). Western blotting is an extremely powerful tool for examining the presence or absence of proteins in a sample and for comparing samples ascertained under different conditions. The major limitation of Western immunoblotting is that it is semi-quantitative.
Enzyme Linked Immunosorbent Assay
Figure 5 Results from polymerase chain reaction (PCR) and real-time PCR analysis. A, this is representative of an ethidium bromide stained agarose gel of PCR products. In the example more product is detected in lane A than lane B. B, this represents a threshold curve of PCR product formation from real-time PCR analysis. In this example the PCR product of sample A is more abundant since it reaches the critical threshold level of fluorescence at an earlier cycle number than sample B.
move more slowly through the gel because they encounter more resistance. Protein bands that result from differential migration are commonly stained for visualisation with Comassie blue or silver and are compared against reference proteins with known molecular weights run on the same gel.
Western immunoblot SDS-PAGE analysis is a powerful technique that allows the examination of global changes in protein levels from biological samples. However, it lacks specificity. Western immunoblot analysis utilises the power of antibody specificity to ascertain information about the levels of a particular protein in biological samples. In this technique, proteins are first separated by SDS-PAGE analysis. Proteins are then transferred to a membrane (nitrocellulose or polyvinylidenefluoride (PVDF)) by applying a current across the gel and membrane in such a way that the proteins migrate from the gel to the membrane (Fig. 6). After the proteins have been transferred, the membrane is incubated with an antibody (primary antibody) raised against the protein of interest. Following incubation with
This technology for analysing proteins also exploits the specificity of antibodies to single proteins. Enzyme Linked Immunosorbent Assay (ELISA) systems use two antibodies, the first captures the protein and the second is used for detection. The first antibody is bound to the bottom of a well (Fig. 7). The protein sample (serum, bronchoalveolar lavage fluid, etc) is added to the well and incubated to allow the specific protein to bind to the antibody. The well is washed extensively so that only the protein that specifically interacts with the antibody is retained. A second antibody that recognises the specific protein and is conjugated with alkaline phosphatase is added to the well. This is also washed extensively and what is retained in the well is an antibody–protein–antibody sandwich. Protein quantification is determined by the relative amount of colour liberated by alkaline phosphatase acting on the substrate. The sample with an unknown concentration of protein is then compared to a standard curve generated from serial dilutions of a known concentration of the protein. ELISA is a quantitative technique commonly employed to analyse cytokine levels in various biological samples including serum, bronchoalveolar lavage and cerebrospinal fluid.
Methods for Analysing Whole Lung The focus of this review has been to discuss molecular biology techniques that are applicable to a whole tissue, a cell or a subcellular compartment but require a homogenate of the sample for analysis. The lung is a complex organ with a host of cell types and an elaborate, physiologically significant architecture. Examining a lung homogenate may not be informative with regard to a disease process that is limited to a small portion of the lung. Therefore, the following molecular biology techniques are useful for examining mRNA and protein expression in the whole lung on histological sections.
BASIC MOLECULAR BIOLOGY
Figure 6
205
The steps involved in a Western immunoblot analysis.
In situ hybridisation
Immunohistochemistry
The principles underlying this are the same as those described for Northern blotting, above. In the case of in situ hybridisation, a radiolabelled oligonucleotide probe is generated to the mRNA of interest. The probe is hybridised against the histological tissue section. The power of this technique is that one can visualise the location of mRNA expression in the context of the whole tissue. In situ hybridisation allows for an estimation of the level and location of expression of mRNA. Since the lung is extremely heterogeneous, in situ hybridisation is a powerful technique since it allows gene regulation to be evaluated over the entire lung. An example of the technique is shown in Fig. 8.
Immunohistochemistry is used to assess the location of specific proteins on a histological tissue section. The technique is similar to Western immunoblot analysis where detection of the protein is determined by using specific antibodies against that protein. The difference is that instead of the protein binding to a membrane, the protein is retained in the cellular architecture found in the histological tissue section. Similar to in situ hybridisation, this technique allows not only the determination of relative protein expression levels but also the distribution of the protein expression. A representative example of immunohistochemistry is shown in Fig. 9.
206
A. P. SENFT AND A. M. LEVINE
Figure 7
The steps involved in an Enzyme Linked Immunosorbent Assay (ELISA) analysis.
SUMMARY Molecular biology techniques have become powerful tools in the practice of medicine. The most immediate examples are the use of ELISA and PCR in routine diagnostic tests. A more interesting and more powerful use of these basic techniques is their use in biological samples from patients. Obtaining information about the underlying mechanisms of human diseases is vital. Information obtained from patients allows the basic research models to be validated as acceptable models for studying specific disease processes. Ultimately, results from translational research may enhance disease prevention and lead to more effective and specific medical treatments.
FURTHER READING: HANDBOOKS OF MOLECULAR BIOLOGY TECHNIQUES The following books are excellent, practical, in-depth resources for protocols to perform the techniques described in this review article. Ausubel F M, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K (eds). Current Protocols in Molecular Biology. New York: John Wiley and Sons, 2005. Bonifacino J S, Dasso M, Harford J B, Lippincott-Schwartz J, Yamada K M (eds). Current Protocols in Cell Biology. New York: John Wiley and Sons, 2005.
BASIC MOLECULAR BIOLOGY
207
Figure 8 In situ hybridisation for surfactant protein-C (SP-C) mRNA in mouse lung. The micrographs depict in situ hybridisation for SPC in adult FVB/N mouse lung (magnification 10X). A depicts the dark-field image to visualise the probe and B depicts the bright-field image of the lung section stained with toludine blue to show the morphological structure. Cellular sites of SP-C expression determined by hybridisation with the anti-sense probe that is complimentary to SP-C mRNA appear as focal clusters (white) throughout the lung parenchyma consistent with the location of alveolar Type II cells (A). In contrast, the bronchiolar epithelium (arrows) is negative for SP-C expression (A and B). The image shown is by courtesy of Dr Stephan Glasser, Cincinnati Children’s Hospital Medical Center.
Figure 9 Immunohistochemistry for pro surfactant protein-C (SP-C) protein in mouse lung. The micrograph shown has been immunohistochemically stained for pro SP-C in 6 week old FVB/N mouse lung (magnification 20X). The arrows point to alveolar Type II cells that stain positive for pro SP-C. The image shown is by courtesy of Dr Susan Wert, Cincinnati Children’s Hospital Medical Center.
208
REFERENCES 1. Rowley AH, Whitley RJ, Lakeman FD et al. Rapid detection of herpessimplex-virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis. Lancet 1990; 335: 440–441. 2. Pauschinger M, Bowles NE, Fuentes-Garcia FJ et al. Detection of adenoviral genome in the myocardium of adult patients with
A. P. SENFT AND A. M. LEVINE
idiopathic left ventricular dysfunction. Circulation 1999; 99: 1348– 1354. 3. Erdman DD, Weinberg GA, Edwards KM et al. GeneScan reverse transcription-PCR assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness. J Clin Microbiol 2003; 41: 4298–4303.
SERIES: BASIC SCIENCE RESEARCH IN RELATION TO THE LUNG
Basic molecular biology Albert P. Senft and Ann Marie LeVine* Divisions of Neonatology and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave.,Cincinnati, OH 45229, USA KEYWORDS Nucleic acids; Proteins; PCR; Immunohistochemistry
Summary Rapid advances in molecular biology over the past 20 years have and will continue to impact on the practice of medicine. Advances in molecular biology are having an immense impact in determining the underlying aetiology of lung disease and its treatment. In this review, basic molecular biology techniques will be discussed with examples of how these techniques are used in clinical practice. ß 2005 Elsevier Ltd. All rights reserved.
INTRODUCTION Molecular biology is a broad area of study aimed at the common goal of understanding the mechanisms of basic cellular function. This review has been written as an introduction to the basic molecular biology techniques used to study DNA, RNA and proteins. The techniques utilised in basic and clinical research as well as the strengths and weaknesses of each technique are described. In addition, a bibliographical list of reference materials for an indepth description of molecular biology techniques has been included.
NUCLEIC ACIDS DNA encodes the molecular template for all molecules necessary for cell function and viability. The nucleotide is the most basic structural unit of DNA and is comprised of deoxyribose sugar, a phosphate group and a purine (adenine and guanine) or pyrimidine (thymine and cytosine) nitrogenous base. DNA is a double helical molecule with two sugar–phosphate strands serving as the backbone of the molecule with the nitrogenous bases projecting toward
* Corresponding author. Tel.: +1 513 636 2995; Fax: +1 513 636 7868. E-mail address: [email protected] (A.M. LeVine). 1526-0542/$ – see front matter ß 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.prrv.2005.06.006
the centre. The two strands are held together by hydrogen bonding, a non-covalent interaction, between the purine and pyrimidine bases. The interaction between the bases occurs with high fidelity (adenine always pairs with thymine and guanine with cytosine). Importantly, the two strands of the DNA molecule can be disassociated and then reassociated with the addition or removal of energy to break the forces of hydrogen bonding. Together these two physical features make DNA a dynamic molecule that allows for its replication and for transcription of encoded genes. The gene is the smallest functional unit of DNA and encodes for proteins. In general, genes are made up of a promoter region and a coding region (Fig. 1). The promoter region of the gene contains short, conserved, defined nucleotide sequences that positively or negatively regulate gene transcription. Gene transcription is a tightly regulated process that is cell-type specific and greatly influenced by the physiological stress placed upon the cell. Gene transcription produces a ribonucleic acid (RNA) template from which a protein is produced. RNA, like DNA, contains a sugar–phosphate backbone and nitrogenous bases. The major differences are that the sugar is ribose, the base uracil is incorporated in place of thymine and the molecule is single-stranded. Total cellular RNA is a mixture of three types: transfer (tRNA), ribosomal (rRNA) and messenger (mRNA), which is the
200
A. P. SENFT AND A. M. LEVINE
Figure 1
A hypothetical gene, primary transcript and final mRNA.
template for protein production. Since mRNA encodes for proteins, this will be the focus of the remaining discussion of RNA. Initial transcription results in the formation of a primary transcript that includes introns, exons and the 30 untranslated region (30 UTR) (Fig. 1). Exons are segments that encode the amino acid sequence of the protein. Introns are intervening sequences that do not encode a protein but guide splicing of the mature mRNA and allow alternative combinations of exons to produce variations of similar proteins. Excision of introns and splicing of exons must occur before mRNA protein translation can occur.
Methods for the Analysis of Nucleic Acids Polymerase Chain Reaction Polymerase chain reaction (PCR) is used to amplify specific DNA sequences from limited biological samples and has revolutionised biomedical research. The technique is based on the following: DNA can be denatured from a doublestranded molecule to a single-stranded molecule by heat. When the single-stranded DNA molecules are cooled they anneal to a double-strand, the process of two single-
stranded DNA molecules annealing occurs with high fidelity (i.e. A always pairs with T and C with G). The polymerase chain reaction is illustrated in Fig. 2. Sequentially, the template DNA is denatured by heating. Specific primers designed for the sequence of interest anneal on the corresponding DNA sequence of the template DNA when the reaction is cooled. DNA polymerase extends the DNA from 50 to 30 building a mirror image of the template strand. At the end of one cycle, two exact copies of template DNA have been formed. Using this technique, DNA can be amplified exponentially. This allows minute amounts of DNA from biological samples to be assayed, making the technique extremely powerful for basic science and clinical research. PCR is a common approach used for the diagnosis of clinical diseases. For example, PCR can be used for the detection of herpes simplex virus in cerebral spinal fluid1 and enterovirus in myocardial biopsy samples.2
Northern blot analysis Northern blot analysis is a sensitive technique for detecting mRNA expression levels. RNA is separated by agarose gel electrophoresis. Since, RNA has a negative charge it
BASIC MOLECULAR BIOLOGY
Figure 2
201
The steps involved in a polymerase chain reaction (PCR).
migrates to the cathode when a current is applied to the gel. Similar to other forms of electrophoresis, mobility of the molecule in the gel is directly proportional to its size. After separation by electrophoresis the RNA is transferred to a nitrocellulose membrane and cross-linked to the membrane by ultra-violet (UV)-irradiation. To determine mRNA levels for a specific gene, a short DNA probe is designed for the specific mRNA of interest. The probe is radiolabelled for detection and hybridised with a nitrocellulose membrane binding the mRNA molecules with a similar sequence. The results of hybridisation are detected by
autoradiography and reveal the number, size and abundance of transcripts (Fig. 3). While this technique is time consuming and requires a significant amount of RNA for analysis, it is still commonly used because it is extremely quantitative and therefore provides an accurate assessment of mRNA levels.
Reverse Transcriptase–Polymerase Chain Reaction Reverse transcriptase–polymerase chain reaction (RT-PCR) is used to assess the level of mRNA expression. As its
202
A. P. SENFT AND A. M. LEVINE
visualised using UV light (Fig. 5A). An important advantage of RT-PCR is that only a small amount of biological sample is necessary to determine the mRNA level. The limitation of this method for determining gene expression is that RT-PCR combined with visualisation using ethidium bromide is only semi-quantitative due to the fact that the PCR reaction is linear only within a range of cycles. While RT-PCR is not well suited for the quantitative comparison of mRNA expression, the technique is useful in screening tissues for the expression of a gene. For example, RT-PCR based assays are used in the clinical setting for the detection of RNA viruses, including respiratory syncytial virus and influenza A virus.3
Real-Time PCR
Figure 3
The steps involved in a Northern blot analysis.
name indicates, a portion of the assay relies on the amplification of a DNA template by PCR. Before PCR amplification, the mRNA is converted into a complimentary DNA (cDNA) molecule. As illustrated in Fig. 4, the enzyme reverse transcriptase is used to make a DNA copy of the mRNA. mRNA is selectively copied by using a primer that binds to the poly A tail of mRNA (not present in the more abundant rRNA and tRNA). Once the cDNA is made, the RNA is degraded by RNAse H. DNA polymerase and T4 DNA polymerase are then used to build the complementary strand. The result is a collection of DNA molecules that represents all the specific mRNA contained in the cell. Similar to the PCR of DNA, primers are designed for a specific gene and, using these primers, the product is exponentially amplified by PCR. The resulting PCR product is subjected to agarose gel electrophoresis to separate the PCR products by size. Ethidium bromide intercalates into the DNA and can be
The technology of real-time PCR developed out of the necessity for a quantitative technique to determine gene expression levels in small samples. Measurement of mRNA for a particular gene by real-time PCR is not fundamentally different from reverse-transcriptase PCR. Both techniques require mRNA initially to be reverse transcribed to a cDNA molecule. The cDNA is then subjected to PCR. The difference lies in the fact that the instrument where the PCR reaction is carried out is both a thermocycler and a detection device. The reaction mix contains a fluorescent probe that intercalates into the DNA during synthesis. At the end of each PCR cycle, the amount of fluorescence is assessed. Based on an algorithm, a threshold level of fluorescence must be reached for an mRNA to be considered expressed and this is expressed as a cycle number (Fig. 5B). mRNA expression is therefore directly related to cycle number; the higher the relative expression the lower the cycle number where the curve crossed the threshold. For example, in Fig. 5B, gene A has a higher expression than gene B because the cycle number, where it crossed the threshold, is lower.
PROTEINS Proteins are the molecules that perform all cellular functions. They are translated from the mRNA by ribosomes. Each amino acid is encoded by at least one triplet nucleotide sequence. Protein translation begins with the codon AUG, which encodes for methionine and is terminated with the stop codons UAA, UGA or UAG. Following translation, proteins are modified (e.g. by glycosylation) and sorted to their appropriate location (e.g. soluble or membranous) by a highly ordered process. With the advent of high-throughput techniques (e.g. microarrays that will be discussed in a future review), examining changes in gene expression under various experimental and pathophysiological conditions has provided a wealth of information and a basis for exploring the
BASIC MOLECULAR BIOLOGY
Figure 4
203
The generation of cDNA from mRNA by the reverse transcriptase reaction.
underlying mechanisms of disease processes. However, changes in gene expression only represent potential changes in protein levels and/or cellular function. Basic approaches to protein analysis are therefore important to understand whether changes in gene expression correlate to changes in protein expression and altered cellular function.
Methods for the Analysis of Proteins Sodium dodecyl sulphate polyacrylamide gel electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique that is used to separate
proteins based on their molecular weight. The detergent SDS has a negative charge and when added to the sample it binds hydrophobic amino acid residues. Proteins bind proportionally to the same amount of SDS. Therefore, the charge to mass of all proteins is equivalent and all SDS– protein complexes are negative. Protein samples are prepared either under reducing conditions where b-mercaptoethanol is added and the sample is boiled, or under nonreducing conditions where b-mercaptoethanol and heat are omitted. The specific sample preparation conditions are determined by experimental necessity. Once the samples have been prepared for electrophoresis, the sample is loaded on the gel and an electric field is applied. The rate of migration of the protein is inversely proportional to the logarithm of its molecular weight, meaning larger proteins
204
A. P. SENFT AND A. M. LEVINE
the primary antibody, the membrane is incubated with an antibody that is conjugated to horseradish peroxidase and is capable of recognising the primary antibody. For example if the antibody against protein X was raised in a rabbit, the second antibody used would be an anti-rabbit horseradish peroxidase conjugated antibody. The highly specific antibody to protein binding is detected using a peroxidase-dependent luminescence generating system followed by exposure to X-ray film. The bands that appear reflect the size and relative amount of the studied protein (Fig. 6). Western blotting is an extremely powerful tool for examining the presence or absence of proteins in a sample and for comparing samples ascertained under different conditions. The major limitation of Western immunoblotting is that it is semi-quantitative.
Enzyme Linked Immunosorbent Assay
Figure 5 Results from polymerase chain reaction (PCR) and real-time PCR analysis. A, this is representative of an ethidium bromide stained agarose gel of PCR products. In the example more product is detected in lane A than lane B. B, this represents a threshold curve of PCR product formation from real-time PCR analysis. In this example the PCR product of sample A is more abundant since it reaches the critical threshold level of fluorescence at an earlier cycle number than sample B.
move more slowly through the gel because they encounter more resistance. Protein bands that result from differential migration are commonly stained for visualisation with Comassie blue or silver and are compared against reference proteins with known molecular weights run on the same gel.
Western immunoblot SDS-PAGE analysis is a powerful technique that allows the examination of global changes in protein levels from biological samples. However, it lacks specificity. Western immunoblot analysis utilises the power of antibody specificity to ascertain information about the levels of a particular protein in biological samples. In this technique, proteins are first separated by SDS-PAGE analysis. Proteins are then transferred to a membrane (nitrocellulose or polyvinylidenefluoride (PVDF)) by applying a current across the gel and membrane in such a way that the proteins migrate from the gel to the membrane (Fig. 6). After the proteins have been transferred, the membrane is incubated with an antibody (primary antibody) raised against the protein of interest. Following incubation with
This technology for analysing proteins also exploits the specificity of antibodies to single proteins. Enzyme Linked Immunosorbent Assay (ELISA) systems use two antibodies, the first captures the protein and the second is used for detection. The first antibody is bound to the bottom of a well (Fig. 7). The protein sample (serum, bronchoalveolar lavage fluid, etc) is added to the well and incubated to allow the specific protein to bind to the antibody. The well is washed extensively so that only the protein that specifically interacts with the antibody is retained. A second antibody that recognises the specific protein and is conjugated with alkaline phosphatase is added to the well. This is also washed extensively and what is retained in the well is an antibody–protein–antibody sandwich. Protein quantification is determined by the relative amount of colour liberated by alkaline phosphatase acting on the substrate. The sample with an unknown concentration of protein is then compared to a standard curve generated from serial dilutions of a known concentration of the protein. ELISA is a quantitative technique commonly employed to analyse cytokine levels in various biological samples including serum, bronchoalveolar lavage and cerebrospinal fluid.
Methods for Analysing Whole Lung The focus of this review has been to discuss molecular biology techniques that are applicable to a whole tissue, a cell or a subcellular compartment but require a homogenate of the sample for analysis. The lung is a complex organ with a host of cell types and an elaborate, physiologically significant architecture. Examining a lung homogenate may not be informative with regard to a disease process that is limited to a small portion of the lung. Therefore, the following molecular biology techniques are useful for examining mRNA and protein expression in the whole lung on histological sections.
BASIC MOLECULAR BIOLOGY
Figure 6
205
The steps involved in a Western immunoblot analysis.
In situ hybridisation
Immunohistochemistry
The principles underlying this are the same as those described for Northern blotting, above. In the case of in situ hybridisation, a radiolabelled oligonucleotide probe is generated to the mRNA of interest. The probe is hybridised against the histological tissue section. The power of this technique is that one can visualise the location of mRNA expression in the context of the whole tissue. In situ hybridisation allows for an estimation of the level and location of expression of mRNA. Since the lung is extremely heterogeneous, in situ hybridisation is a powerful technique since it allows gene regulation to be evaluated over the entire lung. An example of the technique is shown in Fig. 8.
Immunohistochemistry is used to assess the location of specific proteins on a histological tissue section. The technique is similar to Western immunoblot analysis where detection of the protein is determined by using specific antibodies against that protein. The difference is that instead of the protein binding to a membrane, the protein is retained in the cellular architecture found in the histological tissue section. Similar to in situ hybridisation, this technique allows not only the determination of relative protein expression levels but also the distribution of the protein expression. A representative example of immunohistochemistry is shown in Fig. 9.
206
A. P. SENFT AND A. M. LEVINE
Figure 7
The steps involved in an Enzyme Linked Immunosorbent Assay (ELISA) analysis.
SUMMARY Molecular biology techniques have become powerful tools in the practice of medicine. The most immediate examples are the use of ELISA and PCR in routine diagnostic tests. A more interesting and more powerful use of these basic techniques is their use in biological samples from patients. Obtaining information about the underlying mechanisms of human diseases is vital. Information obtained from patients allows the basic research models to be validated as acceptable models for studying specific disease processes. Ultimately, results from translational research may enhance disease prevention and lead to more effective and specific medical treatments.
FURTHER READING: HANDBOOKS OF MOLECULAR BIOLOGY TECHNIQUES The following books are excellent, practical, in-depth resources for protocols to perform the techniques described in this review article. Ausubel F M, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K (eds). Current Protocols in Molecular Biology. New York: John Wiley and Sons, 2005. Bonifacino J S, Dasso M, Harford J B, Lippincott-Schwartz J, Yamada K M (eds). Current Protocols in Cell Biology. New York: John Wiley and Sons, 2005.
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Figure 8 In situ hybridisation for surfactant protein-C (SP-C) mRNA in mouse lung. The micrographs depict in situ hybridisation for SPC in adult FVB/N mouse lung (magnification 10X). A depicts the dark-field image to visualise the probe and B depicts the bright-field image of the lung section stained with toludine blue to show the morphological structure. Cellular sites of SP-C expression determined by hybridisation with the anti-sense probe that is complimentary to SP-C mRNA appear as focal clusters (white) throughout the lung parenchyma consistent with the location of alveolar Type II cells (A). In contrast, the bronchiolar epithelium (arrows) is negative for SP-C expression (A and B). The image shown is by courtesy of Dr Stephan Glasser, Cincinnati Children’s Hospital Medical Center.
Figure 9 Immunohistochemistry for pro surfactant protein-C (SP-C) protein in mouse lung. The micrograph shown has been immunohistochemically stained for pro SP-C in 6 week old FVB/N mouse lung (magnification 20X). The arrows point to alveolar Type II cells that stain positive for pro SP-C. The image shown is by courtesy of Dr Susan Wert, Cincinnati Children’s Hospital Medical Center.
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