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5BBG0205 Molecular Basis of Gene Expression > L10: Methods to study gene expression > Flashcards

L10: Methods to study gene expression Flashcards

1
Q

What are the properties of RNA?

A

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.

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2
Q

How does RNA differ from DNA?

A
  • 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.
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3
Q

3 key differences: DNA vs RNA

A
  • DNA double stranded, RNA single stranded
  • Ribose (not deoxyribose)
  • Uracil instead of Thymine
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4
Q

where can RNA be extracted/isolated from?

A

Whole organism
Cultured cells
Tissue sections
Pathology specimen

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5
Q

What makes RNA vulnerable to degradation?

A

Vulnerable to hydrolysis
Single stranded
Contains ribose

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6
Q

What are Ribonucleases (RNAses)?

A

RNA destroying enzymes
Found in all cells
Don’t require cofactors like DNAses

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7
Q

Where are Ribonucleases (RNAses) found?

A

Found in all cells
Humans shed them in significant quantities

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8
Q

What is the result of the presence of Ribonucleases (RNAses)?

A

High turnover rate

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9
Q

What are the steps involved in RNA isolation?

A

Tissue collection
Homogenization
RNA stabilization
Extraction
Purification/Precipitation

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10
Q

What is Acid GTC-phenol-chloroform extraction?

A

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)

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11
Q

Explain Acid GTC-phenol-chloroform extraction

A
  • 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.
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12
Q

What are the three main types of RNA found in extracted whole human RNA, and what are their approximate percentages?

A

mRNA - approximately 5-10% of total RNA
rRNA - approximately 80-90% of total RNA
tRNA - approximately 5-10% of total RNA

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13
Q

What is Poly A+ selection of Eukaryotic mRNAs and why is it necessary?

A
  • 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)

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14
Q

Why is RNA much less stable than DNA?

A
  • to facilitate precise regulation of gene expression.
  • RNases are ubiquitous & degrade RNA, hence during extraction, they are inhibited with GTC
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15
Q

What technique is commonly employed to isolate mRNA from total extracted RNA, and why?

A
  • 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.
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16
Q

What is Northern analysis (blotting) ?

A

a technique used for separating and analyzing RNA molecules.

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17
Q

What are the steps involved in Northern analysis?

A
  • 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.
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18
Q

How does Northern gel/analysis differ from northern blotting

A

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)

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19
Q

What is Northern blotting commonly used for?

A

to show the relative amount and sizes (length) of specific transcripts (RNAs).

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20
Q

Why is transferring RNA molecules from a Northern gel to a nitrocellulose membrane advantageous?

A
  • makes it easier to probe for a specific transcript of interest
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21
Q

What are some challenges associated with Northern blotting?

A

→ time-consuming.
→ requires a large input RNA sample.

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22
Q

applications of northern blotting?

A
  • 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
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23
Q

What is the PCR and what are its requirements?

A
  • 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)
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24
Q

What are the phases of PCR?

A

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.

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25
Q

What is Reverse Transcription PCR (RT-PCR) and how does it differ from PCR?

A

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.

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26
Q

How does reverse transcriptase work in reverse transcription?

A
  • 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
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27
Q

What are the two approaches to performing RT-PCR?

A

One-step (simultaneous) RT-PCR
Two-step RT-PCR

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28
Q

What are the two steps involved in RT-PCR?

A

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.

29
Q

What are the characteristics of one-step (simultaneous) RT-PCR?

A
  • 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.
30
Q

What are the characteristics of two-step RT-PCR?

A
  • 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.
31
Q

What are the types of primers used in the first step (RT) of RT-PCR, and how do they function?

A
  • 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.
32
Q

What are gene-specific primers, and how do they differ from random hexamers and oligo dT?

A

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

33
Q

What is an example of RT-PCR application?

A

→ 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.

34
Q

How was RT-PCR utilized to study TUCAN gene isoforms?

A

→ 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.

35
Q

What are the differences between Quantitative Real-Time PCR (qPCR), PCR, and RT-PCR?

A

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.

36
Q

What is the process of Quantitative Real-Time PCR (qPCR)?

A
  • 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
37
Q

How is the reaction measured in real-time during Quantitative Real-Time PCR (qPCR)?

A
  • 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.
38
Q

What are the characteristics of SYBR Green I in qPCR?

A

SYBR Green I =asymmetrical cyanine dye
- preferentially binds (intercalates with) dsDNA
- Binding of dsDNA causes a dramatic increase in fluorescence, over 1,000-fold.

39
Q

What are the characteristics of TaqMan assays in qPCR?

A
  • 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.
40
Q

What is the significance of the Ct value in real-time PCR?

A

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.

41
Q

what does a lower Ct indicate?

A
  • Lower Ct values indicate higher target sequence amounts
    -while higher Ct values suggest lower amounts or issues.
42
Q

How can relative expression be calculated in real-time PCR?

A
  • 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.
43
Q

How is relative quantification (RQ) calculated in real-time PCR?

A

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

44
Q

What is a housekeeping gene?

A
  • 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
45
Q

What are some well-known housekeeping genes?

A

GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)
ACTB (Beta-actin)
B2M (Beta-2 microglobulin)
BTUB (Beta-tubulin)

46
Q

What is IRGM?

A
  • gene involved in the immune response
  • variations e.g. deletions, in promoter region of IRGM have been linked to Crohn’s disease susceptibility
47
Q

What is the objective of the qPCR experiment examining IRGM expression in individuals with a promoter variant?

A
  • 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
48
Q

What is the delta delta Ct method used for in qPCR?

A
  • 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.
49
Q

What is the absolute quantification method in qPCR?

A
  • 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
50
Q

How is the absolute quantity of an unknown sample inferred using the standard curve method?

A
  • 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
51
Q

What are some applications of qPCR (Quantitative PCR)?

A

DNA (gene) copy number analysis
Mutation detection
Bacterial load quantification
Viral load quantification
mRNA quantification (gene expression analysis)

52
Q

How does qPCR differ from regular PCR?

A
  • 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)
53
Q

What is the main applications of qPCR?

A

detecting and quantifying the expression level of a gene.

54
Q

What are the two main methods of detection in qPCR?

A

SYBR Green
Taqman probes

55
Q

What are the two main quantification methods in qPCR?

A
  • 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.
56
Q

What is the transcriptome?

A
  • 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.
57
Q

What are some techniques for studying the transcriptome?

A

cDNA libraries
Subtractive screening
Gene expression microarrays
High throughput/massively parallel RNA-sequencing

58
Q

What is a cDNA library?

A
  • a collection of cDNA molecules.
  • derived from the mRNA transcripts of a cell or tissue of interest.
59
Q

How are cDNA libraries typically stored and amplified?

A
  • 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
60
Q

What does a cDNA library represent?

A
  • 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
61
Q

What are the advantages of using a cDNA library?

A

much more stable than storing RNA

62
Q

What are the steps involved in making a cDNA library?

A
  • 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.
63
Q

Why is it necessary to amplify the cDNA library?

A
  • 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.
64
Q

What are some applications of cDNA libraries?

A
  • 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.
65
Q

What is subtractive library screening?

A
  • an early method for analyzing differential gene expression (DGE).
  • allows the detection of genes that are differently expressed between two tissues/states.
66
Q

How does subtractive library screening work?

A
  • 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.
67
Q

What are the two main methods for measuring gene expression on a large scale?

A

Gene expression microarrays
Next-generation sequencing: RNA-sequencing

68
Q

How do gene expression microarrays work?

A

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.

5BBG0205 Molecular Basis of Gene Expression (9 decks)

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