L10: Methods to study gene expression Flashcards
What are the properties of RNA?
RNA = single-stranded nucleic acid molecule.
RNA composed of nucleotides, contains ribose sugar RNA uses U instead of T as one of its bases, pairing with A during transcription.
How does RNA differ from DNA?
- RNA typically shorter-lived & more prone to degradation due to the presence of ribonucleases.
- RNA plays various roles in the cell, including mRNA for protein synthesis, rRNA as part of the ribosome, and tRNA in protein translation.
3 key differences: DNA vs RNA
- DNA double stranded, RNA single stranded
- Ribose (not deoxyribose)
- Uracil instead of Thymine
where can RNA be extracted/isolated from?
Whole organism
Cultured cells
Tissue sections
Pathology specimen
What makes RNA vulnerable to degradation?
Vulnerable to hydrolysis
Single stranded
Contains ribose
What are Ribonucleases (RNAses)?
RNA destroying enzymes
Found in all cells
Don’t require cofactors like DNAses
Where are Ribonucleases (RNAses) found?
Found in all cells
Humans shed them in significant quantities
What is the result of the presence of Ribonucleases (RNAses)?
High turnover rate
What are the steps involved in RNA isolation?
Tissue collection
Homogenization
RNA stabilization
Extraction
Purification/Precipitation
What is Acid GTC-phenol-chloroform extraction?
Phenol-chloroform separates nucleic acids from proteins
GTC denatures proteins, particularly RNAses
Sodium Acetate (pH4) partitions DNA to organic phase
(GTC stands for guanidinium thiocyanate)
Explain Acid GTC-phenol-chloroform extraction
- most common method for RNA isolation.
- involves using a mixture of guanidinium thiocyanate (GTC), phenol, and chloroform.
- GTC denatures proteins, particularly RNAses, ensuring their inactivation
- Sodium acetate (pH 4) used to partition DNA to organic phase.
- After homogenization & centrifugation, phase separation occurs → aqueous phase containing RNA.
- RNA in aqueous phase then precipitated w/ alcohol & centrifuged again to pellet it.
What are the three main types of RNA found in extracted whole human RNA, and what are their approximate percentages?
mRNA - approximately 5-10% of total RNA
rRNA - approximately 80-90% of total RNA
tRNA - approximately 5-10% of total RNA
What is Poly A+ selection of Eukaryotic mRNAs and why is it necessary?
- Poly A+ selection = method to isolate mRNA from other types of RNA in eukaryotic cells.
- utilizes polyA tail present on mRNAs to selectively purify them from other types of RNA.
- necessary because rRNA is highly abundant in cells
- Oligo dT beads commonly used for poly A+ selection.
(note: mRNA is the only RNA that contains polyA tail)
Why is RNA much less stable than DNA?
- to facilitate precise regulation of gene expression.
- RNases are ubiquitous & degrade RNA, hence during extraction, they are inhibited with GTC
What technique is commonly employed to isolate mRNA from total extracted RNA, and why?
- Poly A selection
- mRNA makes up only a small proportion of the total RNA regardless of the tissue source.
- Poly A selection helps in selectively isolating mRNA for studying gene expression.
What is Northern analysis (blotting) ?
a technique used for separating and analyzing RNA molecules.
What are the steps involved in Northern analysis?
- Extracted whole RNA or polyA+ RNA applied to horizontal gel for electrophoresis.
→ resulting pattern of RNA molecules transferred onto a nitrocellulose membrane.
→ RNA immobilized on the membrane then incubated with radioactive/fluorescent probe complementary to gene of interest.
→ visualization of probe reveals presence of RNA and its size, often compared to a size marker or ladder.
How does Northern gel/analysis differ from northern blotting
northen gel/analsyis = first step of northern blotting: → extracted whole RNA or polyA+ RNA applied to horizontal gel for electrophoresis
(don’t go as far as transferring it to another membrane)
What is Northern blotting commonly used for?
to show the relative amount and sizes (length) of specific transcripts (RNAs).
Why is transferring RNA molecules from a Northern gel to a nitrocellulose membrane advantageous?
- makes it easier to probe for a specific transcript of interest
What are some challenges associated with Northern blotting?
→ time-consuming.
→ requires a large input RNA sample.
applications of northern blotting?
- gene expression studies (determine where particular gene is expressed)
- identify the presence of closely related species
- determine size & abundance of RNA
- analysis of RNA processing
What is the PCR and what are its requirements?
- PCR is a method for copying DNA.
- requirements: DNA template, oligonucleotide primers, DNA polymerase enzyme (e.g., Taq polymerase), and nucleotides (A, C, G, T)
What are the phases of PCR?
Lag phase: PCR is initiating but undetectable.
Exponential phase: PCR components are in excess, leading to product doubling.
Linear phase: Efficiency declines with each cycle.
Plateau phase: PCR components have run out.
What is Reverse Transcription PCR (RT-PCR) and how does it differ from PCR?
RT-PCR: adaptation of PCR with some key differences.
→ template that is copied in RT-PCR is mRNA, whereas in PCR, it is DNA.
→ mRNA cannot be amplified directly as no enzyme exists for it.
→ so in RT-PCR, reverse transcriptase is used to convert RNA to DNA before amplification.
How does reverse transcriptase work in reverse transcription?
- Reverse transcriptase makes cDNA from RNA.
- the enzyme originates in RNA viruses called retroviruses.
- Retroviruses use the enzyme to reverse-transcribe their RNA genomes into DNA.
- allows retroviruses to integrate into the host genome and be replicated along with it
What are the two approaches to performing RT-PCR?
One-step (simultaneous) RT-PCR
Two-step RT-PCR
What are the two steps involved in RT-PCR?
1: Reverse transcription (RT) - Reverse transcriptase enzyme used to generate first strand, which is a DNA copy of the RNA.
2: PCR - Taq polymerase is added, and PCR can proceed in the traditional way.
What are the characteristics of one-step (simultaneous) RT-PCR?
- All reagents added at the beginning.
- Both reverse transcription & PCR happen simultaneously.
- Requires specific primer from the outset.
Advantage: Quicker than two-step RT-PCR.
What are the characteristics of two-step RT-PCR?
- RT reaction occurs separately from PCR.
- Can use random priming to make the first strand of cDNA.
Advantage: Can stop after the first step and store cDNA for use in other PCRs later.
What are the types of primers used in the first step (RT) of RT-PCR, and how do they function?
- Random hexamers: A mixture of oligonucleotides representing all possible hexamer sequences [NNNNNN], randomly hybridizing to any RNA sequence.
- Oligo dT: A single-stranded sequence with 12-18 deoxythymidines [TTTTTTTTTTTT], specifically hybridizing to the poly A tail of mRNAs.
What are gene-specific primers, and how do they differ from random hexamers and oligo dT?
Gene-specific primers: Oligonucleotides designed to be perfectly complimentary to specific gene (requiring prior sequence knowledge)
→ enable reverse transcriptase to copy only RNA corresponding to target gene
What is an example of RT-PCR application?
→ Identifying different splice isoforms of TUCAN/CARD8 gene.
→ A DNA change associated w/ inflammatory bowel disease introduces a premature termination codon (STOP) into main 48kDa isoform of the gene (T48) at the 10th amino acid residue.
How was RT-PCR utilized to study TUCAN gene isoforms?
→ Gene-specific RT-PCR was performed on mRNA extracted from blood samples of individuals with & w/o the mutation, as well as other reference tissues.
→ This analysis revealed the presence of at least three additional isoforms of the TUCAN gene.
What are the differences between Quantitative Real-Time PCR (qPCR), PCR, and RT-PCR?
PCR → used to amplify a specific DNA sequence, involving repeated cycles of denaturation, annealing of primers & extension by DNA polymerase.
RT-PCR (Reverse Transcription PCR) → variation of PCR, first converts RNA to cDNA using reverse transcriptase before amplifying the cDNA via PCR.
Quantitative Real-Time PCR (qPCR) → modification of PCR that allows for the quantification of DNA/cDNA in real-time during amplification, typically using fluorescent probes/dyes, provides quantitative data on the starting amount of target DNA or cDNA.
What is the process of Quantitative Real-Time PCR (qPCR)?
- Denaturation: reaction mixture is heated to separate DNA strands.
- Annealing: Primers specific to target DNA sequence anneal to their complementary sequences on single-stranded DNA.
- Extension: DNA polymerase extends primers, synthesizing new DNA strands.
- Fluorescence Detection: During each cycle, fluorescence emitted by binding of fluorescent probe/dye to amplified DNA measured in real-time.
- Quantification: amount of fluorescence correlates with amount of DNA present, allowing for quantification of target DNA
How is the reaction measured in real-time during Quantitative Real-Time PCR (qPCR)?
- Labeling accumulating PCR product: Using fluorescent dye e.g SYBR Green, which binds to all double-stranded DNA using a fluorescent probe/quencher e.g. TaqMan, which is specific to target gene.
- Measuring fluorescence emitted by labeled DNA during each cycle of PCR reaction.
What are the characteristics of SYBR Green I in qPCR?
SYBR Green I =asymmetrical cyanine dye
- preferentially binds (intercalates with) dsDNA
- Binding of dsDNA causes a dramatic increase in fluorescence, over 1,000-fold.
What are the characteristics of TaqMan assays in qPCR?
- TaqMan probes dual-labeled with fluorescent dye & a quencher.
- designed to specifically hybridize with a chosen gene’s PCR product as it accumulates.
-quencher prevents dye from fluorescing while probe is intact. - during each cycle of PCR, bound probe is cleaved by the 5’ nuclease activity of Taq polymerase, releasing dye to fluoresce.
As the PCR product increases, so does fluorescence.
What is the significance of the Ct value in real-time PCR?
Ct value = cycle threshold
- represents cycle number at which sample’s reaction crosses fluorescence threshold, indicating the detection of target nucleic acid
Ct = 28.5 indicates amplification crossed threshold at cycle 28.5.
what does a lower Ct indicate?
- Lower Ct values indicate higher target sequence amounts
-while higher Ct values suggest lower amounts or issues.
How can relative expression be calculated in real-time PCR?
- by comparing Ct values of the target gene in the sample of interest to a comparator sample.
(comparator sample typically expresses the gene at a low level/serves as reference) - by comparing Ct values between sample of interest & comparator, relative expression level of target gene can be determined.
How is relative quantification (RQ) calculated in real-time PCR?
Relative quantification (RQ) can be calculated using the ΔCt method.
ΔCt is the difference in Ct values between the sample of interest (Sample A) and a comparator sample (Sample B).
RQ is calculated as 2 raised to the power of ΔCt.
e.g. if ΔCt = 5, then RQ = 2^5 = 32.
This means that Sample B contains 32 times more of the gene of interest than Sample A.
→ In practice, housekeeping gene often used to control for technical errors & normalize data
What is a housekeeping gene?
- cellular maintenace genes
- constitutively (in all cells) active
- relatively constant level in all tissues & cells
- unaffected by experimental conditions
- acts as endogenous control within sample
What are some well-known housekeeping genes?
GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)
ACTB (Beta-actin)
B2M (Beta-2 microglobulin)
BTUB (Beta-tubulin)
What is IRGM?
- gene involved in the immune response
- variations e.g. deletions, in promoter region of IRGM have been linked to Crohn’s disease susceptibility
What is the objective of the qPCR experiment examining IRGM expression in individuals with a promoter variant?
- to determine if a genetic variation (deletion) in promoter region of the IRGM gene affects its expression.
- to identify Crohn’s disease (CD) patients with & w/o IRGM promoter deletion.
- to collect RNA samples from immune cells (blood lymphocytes) of CD patients w/ & w/o promoter deletion.
- to carry out qPCR of IRGM & the housekeeping gene GAPDH in collected RNA samples.
- to include a comparator sample (placenta) for comparison
What is the delta delta Ct method used for in qPCR?
- used to calculate relative quantification of gene expression in qPCR
- compares change in gene expression between a target sample & a reference sample.
- formula for relative quantification is RQ = 2^(-ΔΔCt), where ΔΔCt represents difference in Ct values between the target & reference samples.
What is the absolute quantification method in qPCR?
- uses a standard curve instead of housekeeping gene.
- involves creating a dilution series of a predefined amount of PCR product or cDNA from gene of interest.
e.g. serial dilutions from 10^8 to 10^1 copies prepared - each dilution used in separate real-time PCR reaction & Ct value calculated for each sample
How is the absolute quantity of an unknown sample inferred using the standard curve method?
- Ct values from dilution series are plotted on a graph, creating a standard curve
- standard curve is used to estimate the absolute quantity of an unknown sample based on its Ct value in qPCR
- In practice, the standard curve samples & the unknown samples are usually run simultaneously on the same qPCR machine
What are some applications of qPCR (Quantitative PCR)?
DNA (gene) copy number analysis
Mutation detection
Bacterial load quantification
Viral load quantification
mRNA quantification (gene expression analysis)
How does qPCR differ from regular PCR?
- QPCR allows real-time monitoring of the PCR reaction.
- quantitative because amount of product is directly related to amount of starting material/template, especially during the exponential phase.
(It is basically PCR..but you follow reaction in real time)
What is the main applications of qPCR?
detecting and quantifying the expression level of a gene.
What are the two main methods of detection in qPCR?
SYBR Green
Taqman probes
What are the two main quantification methods in qPCR?
- Relative Quantification: achieved by comparing target sample to a comparator sample and/or housekeeping gene.
- Absolute Quantification: achieved by comparing to a standard curve derived from a serial dilution.
What is the transcriptome?
- set of all RNA transcripts, including coding and non-coding
- represents the sum total of all gene expression at a given moment in time in a particular cell or tissue.
What are some techniques for studying the transcriptome?
cDNA libraries
Subtractive screening
Gene expression microarrays
High throughput/massively parallel RNA-sequencing
What is a cDNA library?
- a collection of cDNA molecules.
- derived from the mRNA transcripts of a cell or tissue of interest.
How are cDNA libraries typically stored and amplified?
- often inserted into a vector e.g a bacterial plasmid, for easy amplification and storage.
- library can be amplified when needed by allowing the bacteria containing the cDNA library to multiply
What does a cDNA library represent?
- a proportion of the transcriptome from the tissue from which it was collected
- reflects the genes that were being expressed at the time of collection
What are the advantages of using a cDNA library?
much more stable than storing RNA
What are the steps involved in making a cDNA library?
- Isolate mRNA & convert it to cDNA.
- Ligate the cDNA into a restriction enzyme (RE) digested vector, such as a plasmid.
- Transform bacteria with the ligated vectors & select for those with a cDNA insert (cloning).
- Amplify the library by growing bacteria to form colonies of individual clones.
- Each bacterial clone/colony contains one of the genes being expressed in that tissue.
Why is it necessary to amplify the cDNA library?
- to ensure there are enough clones to represent a good selection of genes.
- can require growing bacteria to form colonies of individual clones, which may number greater than 10,000 in practice.
What are some applications of cDNA libraries?
- Studying genes expressed in a tissue.
- Discovering novel genes.
- Studying alternative splicing.
- Comparing genes expressed in different cells, different tissues, and at different stages or under different conditions.
What is subtractive library screening?
- an early method for analyzing differential gene expression (DGE).
- allows the detection of genes that are differently expressed between two tissues/states.
How does subtractive library screening work?
- cDNAs from tissue of interest have adapters added, which are small known sequences.
- these cDNAs then mixed with cDNAs from a control tissue, and similar or same genes are subtracted out.
- this method helps identify genes that are differentially expressed between the tissue of interest & the control tissue.
What are the two main methods for measuring gene expression on a large scale?
Gene expression microarrays
Next-generation sequencing: RNA-sequencing
How do gene expression microarrays work?
Thousands of DNA probes are designed to represent all known genes in an organism.
The probes are fixed to a solid surface (chip) with precisely defined positions.
A cDNA library can be applied to the array.
Any cDNA that hybridizes to a probe on the array will cause a fluorescent signal.
