Molecular Analysis of Cellular Systems

Molecular analysis involves isolating macromolecules (DNA, RNA, protein) and studying their function in vitro and in vivo.
Methods can be applied to single molecules or in a high-throughput manner.
Parallel analyses of DNA, mRNAs, proteins are termed genomics, transcriptomics, proteomics, or -omics in general.

Genetic Engineering – DNA Technology

Enzymes for DNA manipulation (already discussed).
Purification of DNA.
DNA labeling methods.
Polymerase chain reaction (PCR), PCR-based in vitro mutagenesis.
Construction and screening of genomic DNA and cDNA libraries.
Membrane blotting - Hybridization (Southern blot).
DNA-Sequencing

Genetic Engineering – RNA Technology

RNA isolation and cDNA synthesis.
Northern blotting.
Reverse transcriptase -(RT-) PCR and quantitative, real-time PCR (qPCR).

Genetic Manipulation of Complex Systems

Micro RNAs and RNA interference using siRNAs.
Genome editing – CRISPR-Cas9 system

High-Throughput Analyses of Complex Systems

Next Generation Sequencing

Genetic Engineering – Protein Technology

Isolation of proteins from cells, classical methods.
SDS-PAGE and Western blotting.
Immunohistochemistry and Immunofluorescence.
Enzyme-linked immunosorbent assay (ELISA)

Formation of Recombinant DNA (Cloning)

Isolation of DNA Fragment:
- The target DNA fragment to be cloned is isolated from its source using techniques such as PCR or restriction enzyme digestion.
Vector Preparation:
- A vector, often a plasmid, is prepared. Plasmids are small, circular DNA molecules that can replicate independently of the host chromosome.
Treatment with Restriction Enzymes:
- Both the isolated DNA fragment and the plasmid are treated with the same restriction enzymes, resulting in complementary sticky ends.
Ligation:
- The DNA fragment and the plasmid are mixed together and ligase enzyme is added, catalyzing the formation of phosphodiester bonds between the fragments.
Transformation:
- The ligated DNA mixture is introduced into a host organism, typically bacteria, through a process called transformation.
Selection:
- Bacteria are grown on a medium containing an antibiotic to which the plasmid confers resistance. Only bacteria that have taken up the plasmid will survive.
Screening:
- Colonies of bacteria are screened to identify those containing the desired recombinant plasmid. This can involve techniques such as colony PCR or restriction enzyme digestion.
Propagation:
- The selected recombinant bacteria are cultured to amplify the recombinant plasmid DNA.

Cloning of DNA Fragments into Plasmids Mediating Antibiotic Resistance

Purpose:
- This method allows for the insertion of a DNA fragment of interest into a plasmid vector that confers antibiotic resistance to the host organism.
Vector Choice:
- Plasmids are commonly used as vectors due to their small size, ease of manipulation, and ability to replicate independently in bacteria.
Antibiotic Selection:
- Plasmids contain genes that confer resistance to antibiotics, such as ampicillin or kanamycin. Host bacteria containing the recombinant plasmid will survive on media containing the corresponding antibiotic.
Insertion of DNA Fragment:
- The DNA fragment of interest is inserted into the plasmid using restriction enzymes and ligase enzyme as described in the process of forming recombinant DNA.
Transformation and Selection:
- The recombinant plasmid is introduced into host bacteria, which are then grown on media containing the antibiotic. Only bacteria containing the recombinant plasmid will survive, allowing for selection of clones containing the DNA fragment of interest.

Purification of DNA Fragments for Cloning

Purity of DNA is critical for successful cloning.
Low melting point agarose, gel purification, agarose digestion precipitation (0.3M Na-acetate).
Neutralization by high salt and withdrawal of water by the addition of ethanol or propanol is necessary to precipitate DNA or RNA

Transformation of Bacteria

Traditional method involves incubating bacterial cells in concentrated calcium salt solution ( $0.1M CaCl_2$ ).
- The solution makes the cell membrane leaky, permeable to the plasmid DNA (heat shock $42°C$ ).
- Polyethylene glycol (PEG) and Dimethyl Sulfoxide (DMSO)
Newer method uses high voltage to drive the DNA into cells in process called electroporation ( $1.8-2.5kV$ ); 10 to 40kV/cm for bacteria, 0.5 to1kV/cm for mammalian cells

Methods for Amplifying Genes

in vivo
in vitro

Polymerase Chain Reaction (PCR)

To amplify (cyclic) DNA fragments in vitro using a thermostable DNA polymerase.
Technique was first described by Kary Mullis in the 1980s, Nobel Prize 1993.
Material
- Thermostable Polymerase (Thermus aquaticus)
- Oligonucleotide Primers
- DNA template (starting material that has to be amplified)
- Deoxynucleotides (dATP , dGTP , dCTP , dTTP)
- Reaction buber (containing magnesium ions)
Method
- Iterative cycles (20-40) of:
  1. A denaturation step at 94 or $95°C$
  2. Primer annealing to the ssDNA template strands at $55-65°C$
  3. Primer extension at $72°C$

PCR-Based Cloning

Used to insert DNA fragments generated by PCR into a vector for further analysis

Steps Involved in PCR-Based Cloning:

Amplification of DNA Fragment:
- The target DNA fragment is amplified by PCR using primers that introduce restriction enzyme recognition sites or specific sequences for subsequent cloning steps.
Digestion of PCR Product and Vector:
- The PCR product and the cloning vector (typically a plasmid) are both digested with the same restriction enzymes to generate compatible ends for ligation.
Gel Purification:
- The digested PCR product and vector are separated by gel electrophoresis, and the DNA bands of interest are excised and purified from the gel.
Ligation:
- The purified PCR product and vector are ligated together using DNA ligase enzyme. The compatible ends generated by the restriction enzymes allow the DNA fragments to be joined together.
Transformation:
- The ligated DNA mixture is introduced into competent host cells, such as bacteria, through a process called transformation.
Selection:
- Bacteria containing the recombinant plasmid are selected by growing them on a selective medium containing antibiotics to which the plasmid confers resistance.
Screening:
- Colonies of transformed bacteria are screened to identify those containing the desired recombinant plasmid. This can involve techniques such as colony PCR or restriction enzyme digestion.
Propagation:
- The selected recombinant bacteria are cultured to amplify the recombinant plasmid DNA, which can then be isolated for further analysis or manipulation.

Recombinant DNA Technology

DNA-labelling techniques required for detection of interactions of DNA with DNA, RNA or protein
Genomic DNA and cDNA libraries
Membrane blotting and hybridization
DNA sequencing
Labelled Deoxynucleotides
Bacterial DNA Polymerase – Klenow Fragment
- In contrast to DNA Polymerase I, thermostable Taq Polymerase I used in polymerase chain reactions lacks the 3‘ to 5‘ exonuclease activity.

Methods for Labelling DNA

Radioactive Labeling: Incorporating radioactive isotopes into DNA for detection.
Fluorescent Labeling: Attaching fluorescent dyes to DNA strands for visualization.
Biotin Labeling: Adding biotin molecules to DNA probes for abinity-based detection.
Enzymatic Labeling: Using enzymes to incorporate modified nucleotides into DNA.
Chemical Labeling: Modifying DNA with chemical moieties for detection.
Click Chemistry Labeling: Attaching labels to DNA via click chemistry reactions.
PCR-Based Labeling: Incorporating labeled nucleotides during PCR amplification.
In Vitro Transcription: Producing labeled RNA probes through transcription reactions.

Detection of Radio-Labelled DNA

$^{32}P$ -labelled DNA can be used to detect complementary DNA or RNA sequences after hybridization and exposure to X-Ray films

Detection of Non-Radioactive Labels in DNA Fragments

Colorimetric Detection:

Enzymatic reactions with chromogenic substrates can produce color changes for visual detection of labeled DNA.
Examples include alkaline phosphatase-conjugated labels reacting (Redox) with substrates like BCIP/NBT or X-gal for color development.

NTB/BCIP-Reaction

Schematic of the NBT/BCIP reaction:
- When alkaline phosphatase (AP) removes the phosphate group of BCIP (5-bromo-4-chloro-3-indolyl-phosphate) the resulting molecules dimerizes under oxidating conditions to give the blue precipitate (5, 5' -dibromo-4,4'-dichloro-indigo).
- During the reaction with BCIP, NBT (nitro blue tetrazolium) is reduced to its colored form to give an enhanced color reaction.

Enhanced Chemiluminescence

Biotinylated DNA can be detected by binding of streptavidin coupled to horseradish peroxidase.
The peroxidase cleaves substrates such as luminol.
Emission of light can then be detected using a luminometer.

Fluorescent Dyes

GFP is a fluorescent protein with wide use
Photon emitted is at a lower energy (longer wavelength) than the photon absorbed
Thus, the diberence between excitation and emission peaks

Fluorescence-Coupled Antibodies

Two commonly used fluorescent dyes that are covalently bound to antibodies:
- Fluorescein (FITC)
  - Emits an intense green fluorescence when excited with blue light
- Rhodamine
  - Emits a deep red fluorescence when excited with green-yellow light
- Alexa Fluor: 18 diberent fluorescent dyes: emission between 442-775 nm
BrdU labelling- Detection of cells in S-phase
- Bromo-desoxyuridine is incorporated into DNA during S-phase.

PCR-Mediated In Vitro Mutagenesis

4 primers are needed for PCR-mediated in vitro mutagenesis.
2 primers harbor the point mutation (1M, 2M) and are complementary to each other.
2 primers are located at the end of final DNA fragment (1 and 2).
First, 2 PCRs are performed using primers 1 / 1M and 2 / 2M , respectively.
Subsequently, the 2 PCR fragments are purified and mixed together promoting duplex formation between the 2 DNA strands.
Finally, PCR is performed using primer 1 and 2.

Restriction Enzyme Based Cloning

Restriction enzyme-based cloning is the prerequisite for the construction of libraries
DNA libraries represent a complex mixture of genomic or cDNA sequences of cell.

Cutting both vector and DNA fragment with the same restriction enzymes.
Ligating the digested DNA fragment into the vector.
Transforming the recombinant DNA into host cells.
Selecting and screening for colonies containing the desired recombinant vector.
Further analysis and propagation of selected clones.

Genomic and cDNA Libraries – Principle

Libraries are a complete set of DNA clones from a complex population

Construction of cDNA Library

Isolating mRNA and converting it to cDNA.
Fragmenting the cDNA and ligating it into a vector.
Introducing the recombinant DNA into host cells.
Selecting and screening for clones with the desired cDNA inserts.
Amplifying and storing the identified clones for further use.

Screening of DNA Libraries Using Hybridization

Bacteria carrying vectors with inserted DNA fragments
Plate bacteria on petri dish
Bacteria generate visible colonies
Make replica of colonies by pressing nylon filter onto dish
Incubate filter with radioactive DNA probe: probe hybridizes with complementary sequence in fragment
X-ray film detects radioactive colonies on filter
Trace colonies back to original plate

Southern Blot Analysis

Method to detect size and quantity of DNA fragments immobilized to membranes.
The technique was extensively used in the past to establish structure of genes based on the arrangement of restriction sites as well as to analyze position of genes on chromosomes.

Isolation of DNA
Restriction digestion of isolated DNA
Agarosegel electrophoresis
Transfer of separated DNA fragments
Hybridization with specific, labelled DNA-probes
Signal development

DNA-Isolation and Quantification Protein K/Phenol Method

Lysis of cells or tissue using a buber containing SDS
Digestion of RNA with RNAse A/T1
Digestion of protein with Proteinase K/SDS
Extraction of the lysate with Phenol and Phenol/Chloroform
Precipitation of DNA (with Na-Acetate and EtOH on ice or - $20°C$ for 12h)
Solving of DNA in TE (10mM Tris pH=8,0 / 1mM EDTA)

DNA Isolation by Ion Exchange Chromatography

Lyse cells in SDS buber with ProtK and RNAse
Apply to Resin
Elute
Separation of Nucleic Acids on QIAGEN Resin

Quantification and Purity of Isolated Nucleic Acids

Photometric Analysis of Concentration
- $\mu g/ml$ nuclei acid= $OD_{260}$ x calculation factor
- calculation factor (for cuvette with d = 1cm):
  - ds-DNA: 1 $OD_{260}$ Unit = 50 $\mu g/ml$
  - ssDNA: 1 $OD_{260}$ Unit = 35 $\mu g/ml$
  - ssRNA: 1 $OD_{260}$ Unit = 40 $\mu g/ml$
  - ss Oligonucleotides: 1 $OD_{260}$ Unit = 20 $\mu g/ml$
Purity
- Good DNA: $OD<em>{260} / OD</em>{280} = 1.7 - 1.9$
- Good RNA: $OD<em>{260} / OD</em>{280} = 2.0 - 2.2.$

Overview: Southern Blot Analysis

Agarose Gel Electrophoresis Ethidiumbromide (EtBr)-Staining

Intercalates between bases
Binding is proportional to fragment length
EtBr provokes fluorescence upon radiation with UV light – orange color
Sensitivity: 1-2ng DA

Capillary Transfer vs. Electro-Blotting of DNA

Capillary transfer involves passive DNA transfer through capillary action, suitable for small fragments and simple setup.
Electro-blotting uses an electric field for faster and more ebicient transfer, ideal for larger fragments but requires specialized equipment.

Hybridization

Fixation of transferred nucleic acids (UV or $80°C$ ) – oven for hybridization.

Steps:

Pre-Hybridization,
Hybridization,
Washing Steps

Formamide

Formamide is a Critical Component in Hybridizations

Formamide is crucial in hybridizations because it helps destabilize the hydrogen bonds in DNA, facilitating the separation of double-stranded DNA into single strands.
This process, known as denaturation, is essential for hybridization reactions, where single-stranded DNA or RNA probes anneal to complementary sequences.
Formamide also reduces the melting temperature of DNA, allowing hybridization to occur at lower temperatures, which is particularly useful for detecting specific sequences under mild conditions.
Overall, formamide enhances the ebiciency and specificity of hybridization reactions by promoting the accessibility of DNA strands and optimizing the annealing process.

Southern Blot: Detection of Immobilized DNA Using Radio-Labelled Probes

A Southern blot involves the transfer of DNA fragments from a gel onto a solid support membrane, such as nitrocellulose or nylon.
Once immobilized on the membrane, the DNA fragments are exposed to a labeled probe that hybridizes to complementary sequences.
The probe is often radio-labeled, allowing for sensitive detection of the target DNA.
Southern blotting is commonly used for various applications, including gene mapping, gene expression analysis, and detection of DNA polymorphisms.

DNA Sequencing

Sequencing is the process by which you determine the exact order of the nucleotides in a given region of DNA
Dideoxynucleotide sequencing is done through complementary chain synthesis and early termination
The synthesized chains are visualized by methods using:
- Radioactive labels ( $^{35}S$ , $^{33}P$ )
- Nonradioactive labels (fluorescence)

Structure of Dideoxy-Nucleotides

Incorporation of Dideoxy-Nucleotides – Sanger Method

Sanger Sequencing Using Radioactivity

In Sanger sequencing, DNA fragments are synthesized in separate reactions containing chain-terminating dideoxynucleotides (ddNTPs), which halt DNA synthesis when incorporated into the growing DNA strand.
Each reaction contains a mixture of regular dNTPs and a small amount of one specific ddNTP, labeled with a radioactive marker (such as $^{32}P$ ).
As DNA synthesis proceeds, terminated fragments of varying lengths are produced, each terminating with a specific ddNTP.
After synthesis, the DNA fragments are separated by size using gel electrophoresis, resulting in a series of bands representing DNA fragments of diberent lengths.
The gel is then exposed to X-ray film, and the radioactive labels on the DNA fragments cause dark bands to form on the film.
By comparing the positions of these bands with a reference ladder of known lengths, the sequence of the original DNA fragment can be deduced.

Fluorescence-Based Dideoxy Sequencing

In fluorescence-based sequencing the dideoxy- nucleotides are each labeled with diberent fluorescent dyes.
Therefore, the four sequencing reactions can be performed in a single tube, since each fragment, terminated by the reaction, dibers in fluorescence color.
DNA fragments are separated on a capillary sequencer and measured upon detection with a laser provoking fluorescence.

Capillary Sequencing

Capillary sequencing separates fluorescently labeled DNA fragments by size in a capillary tube using electrophoresis.
The resulting chromatogram reveals the sequence of nucleotides in the DNA fragment.

Genetic Engineering – RNA Technology

RNA isolation
Revers transcriptase -(RT-) PCR
quantitative real-time PCR (qPCR)

RNA Isolation

Isolation of RNA from cells works similar to the isolation of DNA using lysis and binding to spin columns.
Since RNA is very unstable, precaution has to be taken, for example utilization of RNAse free water, gloves etc.

RNA Content

One eukaryotic cell contains around 10 pg of total RNA
80 - 85% ribosomal RNA
ca. 15 - 20% tRNA and snRNA, miRNA etc.
ca. 1-5% mRNA

Cultured Cells or Tissue

Prepare Lysate (Lyse cells/homogenize tissue)
Filter Lysate in Filtration Column (Spin 2min)
Bind RNA (Add ethanol, transfer and spin)
Wash Column (Wash and spin 3 times)
Elute Total RNA (Add elution buber and spin 1min= Pure Total RNA

Isolation of Poly A+ RNA

To separate mRNAs from all other types of RNA molecules total RNA is applied to columns filled with Oligo-dT Cellulose, only RNAs containing a poly A tail bind to the resin, which can then be washed to elute pure poly A+ RNA.

Separation of Total RNA and Poly A+ RNA in Agarose Gels

Preparation:
- Total RNA is typically treated with DNase to remove genomic DNA contamination.
- Poly A+ RNA is then isolated using oligo(dT) magnetic beads or chromatography columns, which selectively bind to the poly(A) tails of mRNA molecules.
Gel Loading:
- Total RNA and poly A+ RNA samples are mixed with gel loading buber containing denaturing agents such as formamide and formaldehyde.
- The samples are heated to denature the RNA and then loaded onto an agarose gel.
Electrophoresis:
- The loaded gel is submerged in an electrophoresis buber and subjected to an electric field.
- The negatively charged RNA molecules migrate through the gel towards the positive electrode, with smaller molecules moving faster than larger ones.
Visualization:
- After electrophoresis, the gel is stained with a fluorescent dye that selectively binds to RNA, such as ethidium bromide or SYBR Green. Under UV light, the RNA bands become visible, allowing for visualization and analysis.
Analysis:
- The separated RNA bands can be quantified and compared to assess the integrity and abundance of total RNA and poly A+ RNA samples.
- Additionally, the gel can be transferred onto a solid support membrane for further analysis, such as Northern blotting or RNA sequencing.

Electrophoresis and Transfer of RNA by Northern Blotting

Analogous to Southern Method, however, RNA has to be denatured (secondary structure has to be destroyed)

Single stranded RNA contains many thermostable secondary structures
Separation if these RNAs will cause a smear in agarose gels
Formaldehyde: Blocks hydrogen bonds between bases thereby preventing formation of secondary structures
Denatured RNAs do not have secondary structures
sharp bands in agarose gels

Before electrophoresis RNA has to be denatured first at $65°C$ and then put on ice
Formaldehyde is added during gel electrophoresis preventing re-formation of secondary structures.

Transfer and Hybridization of RNA

Transfer of RNA using high salt solutions (protect RNA from degradation) from
- untreated gels
- oder after 20 min. of incubation with 0.05M NaOH
Staining with methylene-blue to detect RNA transferred to membrane
Detection of specific mRNAs using a $^{32}P$ -labelled cDNA

Semi-Quantitative Reverse Transcriptase (RT) – PCR

Semi-quantitative reverse transcriptase PCR (RT-PCR) involves converting RNA into cDNA, amplifying it with PCR, and analyzing band intensity on a gel to estimate relative RNA transcript abundance.
While less precise than quantitative methods, it obers a rapid and cost-ebective approach for comparing gene expression levels across samples.

Real-Time PCR

Developed in the mid-1990 for the analysis and quantification of the PCR product in real-time
Permits quantification of DNA product during the exponential phase of the PCR reaction
Based on the real time detection of fluorescent dyes
The increase in fluorescence is measured cycle by cycle and directly correlates with the amount of PCR product formed
Real-Time PCR using intercalating dyes
- Denaturation (unbound intercalating dye emits very little fluorescence)
- Hybridization
- Extension (fluorescent signal increases significantly as intercalating dye intercalates the double-stranded DNA)
- Present widely used fluorescent dyes: SYBR Green I, Eva Green, SYBR Green Save
- Increase in fluorescence: ds DNA ~ 2000 times higher than un-bound dye
Typical quantitative PCR (qPCR) result for diSerent concentrations of cDNA
- Low cDNA concentrations: Delayed onset of amplification, higher Ct values
- Mid-range cDNA concentrations: Earlier amplification, lower Ct values
- High cDNA concentrations: Early plateau phase, saturation of reaction
- Comparing Ct values to a standard curve allows quantification of target cDNA
Ct (cycle threshold) in qPCR reactions
- Ct value defines the increase of fluorescence in qPCR reactions above background where the exponential phase of the PCR reaction is initiated.
- Ct values are normalized to reference/housekeeping genes such as TBP (TATA box binding protein which do not change expression under diberent conditions
- $\Delta Ct value = Ct(Target RNA)–Ct(Reference RNA)$

More Features of Real-Time qPCR

Primer Design

Real-time products should be smaller than 250 bp
Primer design with the aid of specific software
- Primer express from Applied Biosystems
- Primer 3 freeware tool
Annealing temperature determined by the software should be used as an initial guide.
Should be verified experimentally e.g. Temperature gradient PCR experiments,

Calculation of Annealing Temperature in PCR Reactions

Wallace-Rule:
- $2(A+T) + 4(G+C) = annealing temperature in °C$
Rule of the thumb:
- Experimental annealing temperature should be 3- $5°C$ below the calculated temperature
- Temperature too low: increased unspecific binding
- Temperature too high: blocks even specific binding to template
„salt adjusted“:
- $Tm = 100,5 + 41 \frac{(C + G)}{(A + C + G + T)} – \frac{820}{(A + C + G + T)} x 16,6 x log10 ([Na^+])$ intracellular concentration cations Na, K etc.): 0,15M
- lowering concentration of cations by a factor of 10 lowers Tm by $17°C$
3‘ ends of primers should be free of:
- Secondary structure
- Repetitive elements
- Palindromes
The forward and reverse primers should have equal GC contents, ideally between 40% and 70%
The binding site on the amplicon should not have extensive secondary structure
Ideal primer concentration should be determined experimentally.
- (0,1 $\mu$ M – 0,5 $\mu$ M end-concentration for both primers, sometimes asymmetric PCR with diberent primer concentrations).
- The optimal primer concentration gives the lowest Ct value (highest sensitivity).

Conditions for Real-Time qPCR Cycling Conditions

Extension (polymerization) time:
- rule of the thumb: amplicon length divided by 25
- Typical Taqman hydrolysis probe Real time PCR protocol
- 1x: 10 min $95°C$ ; 30-40x: 20 sec $95°C$ 45 sec $60 °C$ 10 sec $4°C$
Control Reactions
- No template (negative) control
- Positive control: to avoid false negative results
- Standard concentrations of template for absolute quantification

TaqMan qPCR Reaction

Primer and Probe Design:
- Gene-specific primers and a fluorescent probe are designed to amplify the target sequence.
- The probe contains a fluorophore and a quencher, which are in close proximity to each other, resulting in quenching of the fluorescent signal.
PCR Amplification:
- The qPCR reaction mixture contains the target DNA template, gene- specific primers, fluorescent probe, Taq polymerase, and dNTPs.
- During PCR cycling, the primers anneal to the target sequence, and Taq polymerase extends the DNA strand, displacing the probe.
- This results in separation of the fluorophore from the quencher, leading to an increase in fluorescent signal.
Fluorescence Detection:
- Fluorescent signal is monitored in real-time during PCR cycling.
- The increase in fluorescence is proportional to the amount of PCR product generated.
- The cycle threshold (Ct) value, which represents the cycle number at which the fluorescence signal crosses a predetermined threshold, is used to quantify the initial amount of target DNA in the sample.
Data Analysis:
- The Ct values are compared to a standard curve generated from known concentrations of a reference sample to determine the initial concentration of the target DNA.
- This provides quantitative information about the abundance of the target sequence in the sample.
TaqMan qPCR obers high specificity, sensitivity, and quantification accuracy, making it a widely used technique for gene expression analysis, SNP genotyping, and microbial quantification.

qPCR: ΔΔCt Method for Relative Quantification of Gene Expression

Selecting Reference Genes:
- Identify suitable reference genes (housekeeping genes) that show stable expression across experimental conditions.
- These genes are used to normalize the expression levels of the target gene.
Calculate Ct Values:
- Perform qPCR to determine the Ct values for both the target gene and the reference gene(s) in each sample.
- Ct values represent the cycle number at which the fluorescent signal crosses a predetermined threshold.
Calculate ΔCt:
- Calculate the ΔCt value for each sample by subtracting the Ct value of the reference gene from the Ct value of the target gene.
- This normalizes the target gene expression to the expression of the reference gene.
Calculate ΔΔCt:
- Calculate the ΔΔCt value by subtracting the ΔCt value of a reference condition (control or calibrator sample) from the ΔCt value of each experimental condition.
- This represents the diberence in target gene expression between the experimental and reference conditions.
Calculate Fold Change:
- Calculate the fold change in gene expression using the formula
- $2^{-(\Delta\Delta Ct)}$ .
- This provides a relative measure of the change in gene expression between the experimental and reference conditions.
- A fold change of 1 indicates no change in expression, while values greater than 1 indicate upregulation and values less than 1 indicate downregulation.
The ΔΔCt method allows for robust and reliable comparison of gene expression levels across diberent experimental conditions, while minimizing variability introduced by factors such as RNA quality and PCR ebiciency.

Genetic Manipulation of Cellular Systems and High Throughput Analyses

Micro RNAs and RNA interference using siRNAs
Genome editing – CRISPR-Cas9 system
Next generation sequencing

Mammalian miRNA Biogenesis and Function

MicroRNAs are initially transcribed by RNA polymerase II as long primary transcripts known as primary-miRNAs (primiRNAs).
MicroRNA hairpins are recognized and excised from primary (pri)-miRNAs in the nucleus by the microprocessor complex, which includes the RNase III enzyme drosha and its binding partner DGCR8 to form pre-miRNAs of ~70nt.
Pre-miRNAs are rapidly exported to the cytoplasm by the nuclear export factor exportin 5 which uses Ran-GTP as a cofactor.
Further cytoplasmic processing by dicer, a second RNase III enzyme, produces an ~18–24 nucleotide duplex.
This fully processed duplex is incorporated in an ATP-independent manner into a large protein complex known as the RNA- induced silencing complex, or RISC.
With help of Argonaut one strand of the miRNA is discarded.
Other strand guides RISC to target RNA provoking RNA degradation or translational inhibition.

Small Interfering RNAs – siRNAs

siRNAs are usually double-stranded RNA molecules (20-25 bp), only phosphorylated at the 5’ end and 2 nucleotide overhang.
In vivo siRNAs are generated as a defense mechanisms against viruses with a RNA genome, but also play a role in the regulation of gene expression
In vitro siRNAs are transfected into cells to specifically downregulate transcript levels of a target gene or to block translations of its mRNA.
Downregulation of gene expression after siRNA treatment can be analyzed by qPCR and/or Western blotting.

Genome Editing – CRISPR-Cas9 System

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) – Cas 9 (endonuclease) system, is a adaptive mechanisms in bacteria to defend against viral infection.
DNA of phages can be integrated by the action of Cas proteins into CRISP.
Integration of the sequences into the bacterial DNA confers immunity against the phage.
System can be used to delete or integrate DNA sequences into specific target regions of the genome.

Steps:

An RNA complementary to the target gene sequence is fused to a guidance RNA.
After transfection of a Cas9 plasmid and the plasmid harboring the guidance RNA target RNA fusion into the cell, a Cas 9 protein – RNA complex is formed.
The guidance RNA brings Cas9 to its complementary target DNA sequence resulting in double strand breaks performed by Cas9.
Upon repair of breaks by non-homologous end joining, sequences are deleted resulting in inactivation of the gene
Alternatively, DNA sequence can be inserted by homologous recombination (knock-in)

Whole Genome Sequencing

Multiple copies of the genome are cut into pieces, sequenced and assembled

Cut many genome copies into random fragments
Sequence each fragment
Overlap sequence reads
Overlap contigs for complete sequence

Next Generation Sequencing

Steps:

Template preparation (randomly breaking genomic DNA to less than 1 kB, ligation of adaptors containing universal priming sites, immobilization to solid surface or in emulsion)
Sequencing (Cyclic Reversible Termination Method)
Imaging

Template Preparation:

Clonally Amplified Templates (Roche, Illumina)
Single-Molecule Templates (Helios BioSciences)

Cyclic Reversible Termination:

incorporation of fluorescent dye, termination, cleavage: removal of dye and terminating group, regeneration of 3´OH-group (incorporate all four nucleotides, each label with a diberent dye – wash, four-color imaging – cleave dye and terminating groups, wash)

Genetic Engineering – Protein Technology

Isolation of proteins from cells, classical methods
SDS-PAGE and Western blotting
Immunohistochemistry and Immunofluorescence
Enzyme-linked immunosorbent assay (ELISA)

Protein Extraction from Cells

Proteins can be isolated by using detergens (SDS, Triton-X, NP-40), high salt