Differential Display and Its Applications PDF

Differential Display and Its Applications  Differential Display  A powerful technique for analyzing differences in gene expression.  Until 1992, subtractive hybridization was the only method that could isolate differentially expressed genes.  In 1992, Liang and Pardee developed a new PCR...

Differential Display and Its Applications  Differential Display  A powerful technique for analyzing differences in gene expression.  Until 1992, subtractive hybridization was the only method that could isolate differentially expressed genes.  In 1992, Liang and Pardee developed a new PCR-based technique called Differential Display (DD).  Focused on detecting differentially expressed genes among nearly 15000 mRNA sequences in mammalian cells.  First described to compare messages that differ between normal and tumorigenic cells.  Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions.  Why Measure Gene Expression?  Assumption that more abundant genes/transcripts are more important  Assumption that gene expression levels correspond to protein levels  Assumption that a normal cell has a standard expression profile/signature  Changes to that expression profile indicate something is happening  Gene expression profiles represent a snapshot of cellular metabolism or activity at the molecular scale  Gene expression profiles represent the cumulative interactions of many hard to detect events or phenomena  Gene expression is a “proxy” measure for transcription/translation events  Introduction  The human genome has been completely sequenced ,of the estimated 30,000 genes embedded in our genome, only a fraction of them, perhaps 10–15%, are „„turned on‟‟.  The study of expressed genes has had a great impact on biological research.  The challenge moves from identifying the parts of the human genome, to understanding their function in health and disease, a field that has been called "functional genomics" or "post-genomic area". High Throughput Measurement Genomics DNA Easy Transcriptomics RNA Ease of Proteomics protein measurement metabolite Metabolomics, Phenomics (etc.) phenotype Hard Measuring Gene Expression  Hybridization: Northern/Southern Blotting DNA Microarrays or Gene Chips  Sequencing:  Serial analysis of gene expression (SAGE). RNA-Sequencing  Polymerase chain reaction (PCR)-based approaches: RT-PCR (real-time PCR) Differential display PCR(DD-PCR) Northern Blotting  The northern blotting is used to study gene expression by detection of RNA.  The Northern blot, also known as the RNA blot  Workflow: I. mRNA is first separated on an agarose gel II. transferred to a nitrocellulose filter III. denatured and hybridized with 32P labelled complementary DNA IV. probe will bind to membrane and form double stranded DNA-RNA hybrid V. The final step is to intensity of band indicates Advantages and Disadvantages  Advantages: Inexpensive, quantitative method of measuring transcript abundance Well used and well understood technology Use of radioactive probes makes it very sensitive  Disadvantages: Relies on radioactive labelling – “dirty” technology Quality control issues “Old fashioned” technology, now largely replaced by microarrays and other technologies Microarray  Essentially high throughput that uses Cy3 and Cy5 fluorescence for detection  The microarrays technology is aimed to measure the gene expression profile of cell.  Microarrays are hybridized with labeled cDNA synthesized from a mRNA-sample of some tissue.  The intensity of label (radioactive or fluorescent) of each spot on a microarray indicates the expression of each gene.  One-dye arrays (usually with radioactive label) show the absolute expression level of each gene.  Two-dye arrays (fluorescent label only) can indicate relative expression level of the same gene in two samples that are labeled with different colors and mixed before hybridization.  Advantages and Disadvantages  Advantages:  High throughput, quantitative method of measuring transcript abundance  Avoids radioactivity (fluorescence)  Kit systems and commercial suppliers make microarrays very easy to use  Uses many “high-tech” techniques and devices  Disadvantages:  Relatively expensive  Microarrays are subject to cross hybridisation bias  Quality and quality-control is highly variable  Analysis and interpretation is difficult  Measurements are relative to a control specimen.  SAGE  SAGE = Serial Analysis of Gene Expression  Principle is to convert every mRNA molecule into a short (10-14base), unique tag.  Based on serial sequencing of 10 to 14-bp tags that are unique to each and every gene.  SAGE is a method to determine absolute abundance of every transcript expressed in a population of cells  Because SAGE does not require a preexisting clone (such as on a normal microarray), it can be used to identify and quantitate new genes or transcript as well as known genes. Advantages and Disadvantages  Advantages: Very direct and quantitative method of measuring transcript abundance Open-ended technology Built-in quality control: e.g. spacing of tags & 4-cutter restriction sites  Disadvantages: Expensive, time consuming technology - must sequence >50,000 tags per sample Most useful with fully sequenced genomes (otherwise difficult to associate 15 bp tags with their genes) 3’ ends of some genes can be very polymorphic RNA-Seq  Same concept as sequencing ESTs and counting SAGE tags, but does not stop at short segments and tags  RNA-seq describes a collection of experimental and computational methods to determine the identity and abundance of RNA sequences in biological samples.  Is being sequenced the cDNA from the mRNA component.  Sequencing of whole transcriptome of a sample (NGS),and comparing it against the whole transcriptome of another sample. Applications of RNA-seq  RNA-seq can be applied to a broad range of scientific questions such as:  Gene expression profiling between samples;  Study of alternative splicing events associated with diseases;  Allele-specific expression,  Disease-associated single nucleotide polymorphisms (SNPs)  Gene fusions to understand, e.g. disease causal variants in cancer.  Single-cell RNA-seq has recently emerged as a way to study complex biological processes, cellular heterogeneity, and diversity, especially in the stem cell biology and neuroscience fields. Advantages and Disadvantages  Advantages:  Very direct and quantitative method of measuring transcript abundance  Higher sensitivity for genes expressed either at low or very high level.  No prior knowledge of genome required  Gives detail about novel transcribed regions, alternative splicing and allele-specific expression.  RNA-seq is considered unbiased.  Open-ended technology  Disadvantages:  Expensive equipment (instruments are >$500,000)  Expensive to run (at least for now)  Amplification steps can distort the balance between abundant and rare RNA species  Selection and hybridization methods may introduce artifacts  Software is still evolving/improving Reverse Transcriptase PCR  Two kinds of “RT-PCR” - confusing 1. One uses reverse transcriptase (RT) to help produce cDNA from mRNA 2. Other uses real time (RT) methods to monitor PCR amplification  Real Time PCR incorporates the ability to directly measure and quantify the reaction while amplification is taking place.  RT (Real Time) PCR is a method to quantify mRNA and cDNA in real time  A quantitative PCR method  Measures the build up of fluorescence with each PCR cycle  Generates quantitative fluorescence data at earliest phases of PCR cycle when replication fidelity is highest. Advantages and Disadvantages  Advantages:  Sensitive assay, highly quantitative, highly reproducible  Considered “gold standard” for mRNA quantitation  Can detect as few as 5 molecules  Excellent dynamic range, linear over several orders of magnitude  Disadvantages:  A little Expensive (instruments are >$150K, materials are also expensive)  Not a high throughput system (10’s to 100’s of genes – not 1000’s)  Can pick up RNA carry over or contaminating RNA leading to false positives Differential Display  A popular method for isolating novel genes in a variety of biological systems including carcinogenesis, hormone regulation, plant biology and neurobiology.  Basic idea:  Run two RNA (cDNA) samples side by side on a gel.  Excise and sequence bands present in one lane, but not the other  The trick:  Reduce the complexity of the samples by making the cDNA with primers that will prime only a subset of all transcripts  Differential display PCR  DD takes advantage of three of the most simple, powerful and commonly used molecular biological methods: RT-PCR cDNA cloning DNA sequencing gel electrophoresis  The DD methodology, also referred to as DDRT-PCR or DD-PCR in PCR nomenclature.  A researcher will study at least two samples, but many more can be studied if the experiment suggests so, one of the beauties of the technology. Differential Display-PCR (DDRT-PCR)  The differential display is a variation of standard PCR, allowing the amplification of a large population of fragments, rather than the specific amplification of one band.  DDRT-PCR displaying subsets of mRNAs from different cell types or tissues , can be compared and used for isolation of the genes of interest.  The general strategy for DD-PCR consists of the combination of: 1) reverse transcription using an anchor primer. 2) performing PCR with the anchor primer and an arbitrary primer. 3) separation of the PCR product by electrophoresis and visualization. Differential Display-PCR (DDRT-PCR)  Anchor Primers  Anchor primers, such as oligo(dT) primers, are designed to bind specifically to the poly(A) tails of eukaryotic mRNAs.  The number of anchor primers can vary based on experimental design.  Traditionally, 12 anchor primers were suggested to cover different mRNA populations effectively.  However, more recent methods have shown that using as few as 3 to 4 anchor primers can yield comparable results while reducing the number of reactions Anchor primers Differential Display-PCR (DDRT-PCR)  Hexamer Primers  Hexamer primers are short oligonucleotides that consist of all possible combinations of six nucleotides (A, T, C, G).  Given that there are 4^6 = 4,096 possible combinations for hexamer sequences.  However, in practice, a smaller subset is often used due to factors like cost and the specific requirements of the experiment.  For instance, studies have reported using around 76 random hexamer primers in combination with other primers to achieve effective cDNA synthesis and amplification. arbitrary primer Differential Display Effective Modifications  Reamplification Clone screening Precloning Vector Sequences Protocols  Screening Large Screens Use of cDNA Instead of RNA  Detection Radioactivity Staining FluorescenceChemiluminescence Reamplification:  Clone screening involves the identification of specific cDNA clones from a library through DDRT-PCR.  This step is crucial for confirming the presence of the desired transcripts and for further functional analysis.  Methodology I. Isolation of cDNA: After initial amplification, the cDNA fragments of interest are excised from agarose gels and purified. II. Ligation into Vectors: The purified cDNA is then ligated into suitable cloning vectors. III. Transformation: The ligated vectors are introduced into competent bacterial cells (e.g., E. coli). IV. Screening: Transformed cells are plated on selective media, and colonies are screened using :  Colony PCR: To amplify inserts directly from colonies.  Hybridization: Using labeled probes specific to the target cDNA.  Sequencing: To confirm the identity of the clones.  Reamplification:  Precloning refers to the preparation steps taken before cloning cDNA fragments into vectors.  This ensures that the fragments are suitable for insertion and subsequent amplification.  Methodology I. Purification: After DDRT-PCR, cDNA products are purified to remove residual primers, nucleotides, and enzymes. II. Restriction Enzyme Digestion: cDNA be digested with restriction enzymes to create compatible ends for ligation into vectors. III. End Repair or A-tailing: Depending on the vector system used, modifications like end repair or adding A-tails may be performed to facilitate ligation. Benefits  Increases the efficiency of cloning by ensuring that cDNA fragments are appropriately prepared.  Reduces background noise from non-specific products during cloning. Reamplification:  Vector sequences protocols involve guidelines for selecting and utilizing plasmids or other vectors for cloning purposes in DDRT-PCR applications.  Methodology I. Selection of Vector: Choose a vector based on factors such as:  Type of promoter (e.g., constitutive or inducible).  Selection markers (e.g., antibiotic resistance).  Compatibility with downstream applications (e.g., expression systems). II. Ligation Protocol: Follow specific protocols for ligating cDNA into vectors, including:  Preparing a molar ratio of insert to vector (typically 3:1).  Incubating with T4 DNA ligase under optimal conditions. III. Transformation Efficiency: Use competent cells with high transformation efficiency to maximize yield. Screening  Large screening techniques allowing researchers to efficiently identify differentially expressed genes across multiple samples.  Methodology I. 96-Well Technologies: This approach significantly increasing throughput. Each well can contain a different sample or condition, enabling parallel analysis of gene expression. II. Hybridization Filter Arrays: These arrays allow for the simultaneous hybridization of multiple cDNA samples against a set of probes on a membrane. This method provides a rapid means of confirming differential expression by detecting specific sequences. III. Reverse Northern Assays: involving hybridizing labeled cDNA to a membrane containing immobilized RNA. This method provides a quick way to assess gene expression levels.  Detection I. Radioactivity:[a-35S]dATP was originally chosen  Performed with end labeled primers result in more intense labeling of small fragments.  Disadvantage: increased physical protection is required II. Staining: silver staining  Allows direct visualization and isolation of the product  Disadvantage: Less sensitive, procedures are difficult to control, and results are inconsistent III. Fluorescence: safety, stability of reagents, low cost and easy disposal, and high throughput  Useful when the purpose of the experiment is to screen a large number of mRNA species IV. Chemiluminescence (Nonradioactive method): Digoxigenin (DIG)- labeled cDNA probes can be used in hybridization filter arrays.  a sensitive method without the use of radioactivity.  Chemiluminescent signals can be easily quantified and visualized, enhancing the reliability of results.  Advantages of DDRT-PCR  It is a sensitive technique.  No special equipment is needed.  Rapidity and simplicity of assays, increased sensitivity, reproducibility.  Possibility to perform an effective search starting with very small amounts of RNA (small quantities of RNA).  Ability to identify differentially expressed genes in more than one population.  Ability to compare several cell populations or variables simultaneously.  No prior information about the mRNA is needed. Disadvantages of DDRT-PCR  Presence of many false positive results with a frequency of greater than 70%. binding of primers to contaminated DNA The binding of primers to a non-specific sequence at low annealing temperature  Not useful in single mode as the candidate gene is identified based on comparative expression mode.  it is insufficient to cover all the genes expressed in a tissue.  Not easily automated or scaled-up.  It would be fairly expensive to get hundreds of differentially expressed clones. Potential Applications  DDRT-PCR has been used to answer many biological questions in mammalian and other nonfungal systems.  However,the same biological enigmas exist in medical mycology.  Many of the approaches reported for mammalian systems are easily adaptable to fungal systems.  A representative but by no means exhaustive sample of potential DDRT-PCR applications is described below. Potential Applications  Cell Differentiation  Cell Cycle and Life Stages  Cell Activation and Signaling  Markers and Mutations  Drug Resistance and Targets for Drugs  Nutritional and Environmental Stress  Low-Abundance Samples and In Vivo-Expressed Genes  Pathogenesis  Microbial Pathogenesis (Nonfungal)  Cell Differentiation  A common application of DDRT-PCR is to identify genes that are regulated during differentiation or development in-cluding: hematopoiesis neuronal cell differentiation Senescence adipocyte differentiation  The advantage of DDRT-PCR is that several stages of development or numerous cell populations undergoing similar differentiation pathways can be compared at the same time. Cell Cycle and Life Stages Cell Cycle Analysis  DDRT-PCR can be employed to investigate gene expression changes that occur during specific phases of the cell cycle (G1, S, G2, and M phases).  By comparing RNA samples isolated from cells at different stages, researchers can identify genes that are upregulated or downregulated as cells progress through the cycle.  DDRT-PCR can identifiy Cell Cycle Regulators  For example, genes related to cyclins, cyclin-dependent kinases (CDKs), and checkpoint proteins can be identified, providing insights into how these factors influence cell proliferation and division.  DDRT-PCR can reveal Cell Cycle Arrest The technique can also reveal changes in gene expression associated with cell cycle arrest due to stress or damage, helping to elucidate mechanisms of cellular response to adverse conditions.  Cell Cycle and Life Stages  Developmental Stages  DDRT-PCR is particularly useful for studying gene expression during different life stages, such as embryonic development, maturation, and aging.  Stress Responses  DDRT-PCR allows for the identification of stress-responsive genes across different life stages or cell cycle phases (e.g., oxidative stress, nutrient deprivation).  Cancer Research  DDRT-PCR can help identify oncogenes or tumor suppressor genes that show differential expression between cancerous and normal cells at various stages of tumor progression.  Immune Response  The technique can be applied to study how immune cells change their gene expression profiles during activation or differentiation throughout an organism's life stages. Cell Activation and Signaling  DDRT-PCR has been applied in many laboratories to identify genes involved in signal cascades.  The importance of signaling cascades is to transfer an environmental signal through a series of proteins to eventually activate transcription and subsequent cellular functions.  These targets have often been identified by comparing cells before and after stimulation of the cell by various factors.  The studies have included stimulation of cells with cytokines, anti-body, and/or ligands.  Markers and Mutations  A potential application of DDRT-PCR is the identification of cell surface or diagnostic markers including cell, tissue, or pathogen-specific molecules.  Specific studies have included identifying metastatic genetic markers in:  prostate cancer,  potential probes for:  rhabdysarcomas  a Mycoplasma fermentas species specific  marker cell surface markers for fibroblasts  molecular regional markers in freshwater planaria  Markers and Mutations  Similar to identifying cell specific markers is the identification of mutations.  Since PCRs can be compared side by side on sequencing gels, small differences in sequence can be easily detected.  DDRT-PCR was used to screen for genes involved in DNA repair by comparing Fanconia anemia and normal fibroblasts.  DD identified deletions in the 3‟ UTR of the human a tropomyosin (TPM1) gene.  The deletions were 5 and 11 bp long in a tandem repeat, a region that has regulatory effects in cell growth and tumor suppression in transformed cells.  It is significant that DDRT-PCR can identify deletions in a single allele. Drug Resistance and Targets for Drugs  A use for DDRT-PCR is to identify new drug targets and the mechanisms of drug resistance or toxicity.  The difference between resistant and sensitive cells may also be analyzed in the absence of drug in order to provide insight into why a cell is sensitive or resistant to drug action.  The effect of drugs can also be examined in vivo by feeding animals the drug and then assessing differential expression in target tissues. Nutritional and Environmental Stress  DDRT-PCR affords a new tool to identify effects resulting from the absence of specific nutrients.  This is often approached by studying tissues isolated from nutrient-deficient versus nutrient fed animals, e.g., those fed copper and lithium.  Another beneficial approach of DDRT-PCR would be the ability to identify genes that are expressed only under certain environmental conditions in vivo. Low-Abundance Samples and In Vivo- Expressed Genes  In many instances, only a small number of cells can be harvested and consequently the quantity of mRNA is limiting.  DDRT-PCR was successfully performed on mRNA isolated from air-dried, snap-frozen mammalian tissue which had been adhered to a cryostat chuck.  These methods may be useful in identifying microbial genes expressed in vivo if clinical samples are collected properly.  Pathogenesis  DDRT-PCR can serve a dual purpose by providing information about the:  pathogenesis of an illness  providing novel markers for diagnosis and therapy.  The identification of differentially expressed genes aids in the understanding of the molecular mechanisms of pathogenesis.  DDRT-PCR has been successfully used to identify candidate genes in disease.  Numerous laboratories have used this approach to gain insight into:  cardiovascular disorders  neurological disorders  cancer  chronic idiopathic fatiguing illness