Methods to Study Gene Expression (II)

Question 1

Northern blotting

Answer 1

• RNA is separated by agarose gel electrophoresis
• Transferred onto nitrocellulose membrane
• Blotting with radioactive DNA probe specific to gene(s) of interest
• Autoradiography

Question 2

S1 Nuclease Protection Assay

Answer 2

• RNA is hybridized to radiolabeled, single-stranded DNA (target- specific)
• Digestion with S1 nuclease (cleaves only single-stranded DNA)
• Gel electrophoresis + autoradiography

Question 3

Real-time quantitative PCR

Answer 3

• RNA
• First-strand cDNA
• PCR + fluorescent dye

Question 4

qPCR Variants

Answer 4

• SYBR-Green: A dye that binds to double-stranded DNA.
• TaqMan: Uses a fluorescently labeled probe that is cleaved during amplification.
• Droplet Digital PCR (ddPCR): A more precise, absolute quantification method that partitions the sample into thousands of tiny droplets.

Question 5

In situ hybridization

Answer 5

1. Detection of transcripts in tissue sections
2. Probe: complementary DNA or RNA
3. Detection:
   * autoradiography
   * fluorescence microscopy
   * immunohistochemistry

Question 6

Green Fluorescent Protein (GFP)

Answer 6

Fluorescent proteins, such as Green Fluorescent Protein (GFP), are used as tags to visualize where and when a gene is expressed, or to track the resulting protein in vivo.

• The gene encoding the fluorescent protein can be fused to a gene of interest or placed under the control of a specific gene’s promoter.
• The resulting expression is visualized using fluorescence microscopy.
• Example: The Brainbow technique uses combinatorial expression of fluorescent proteins in the nervous system to color-code individual neurons, allowing complex wiring patterns to be studied.

Question 7

Reporter Gene Assays

Answer 7

Reporter gene assays are used to study the activity of a gene’s promoter (the regulatory sequence) by fusing it to a reporter gene. The activity of the reporter gene product is easily measurable and serves as a proxy for the promoter’s activity.
Common Reporter Genes:
* Firefly luciferase
* Renilla (‘sea pansy’) luciferase
* GFP and its variants
* Chloramphenicol acetyltransferase (CAT)

Question 8

NanoLuc

Answer 8

• Derived from a shrimp luciferase (Oplophorus gracilirostris).
• It is smaller (19 kDa vs. Firefly’s 61 kDa or Renilla’s 36 kDa). * It is approximately 100x brighter than firefly or Renilla luciferase, making it extremely sensitive.
• It is highly useful for fusion proteins due to its small size.

Question 9

“Dual Glo“ Luciferase Assay System

Answer 9

Assay systems like the “Dual Glo” Luciferase Assay System allow for the simultaneous measurement of two different reporter genes (e.g., Firefly and Renilla) for internal control and normalization.

Step 1: Firefly Luciferase:
* Substrate: Beetle Luciferin (+ ATP, Mg²⁺, O₂).
* Output: Light (Flash) proportional to the experimental gene’s expression.

Step 2: Renilla Luciferase:
* Trigger: Add Quenching Reagent to stop Firefly light and start Renilla.
* Substrate: Coelenterazine (+ O₂).
* Output: Light proportional to the control gene’s expression.

The Goal (The “Why”): * Normalization: Renilla acts as an internal control to account for differences in cell number or transfection efficiency.
* Calculation: Result = (Firefly Activity) / (Renilla Activity).

Question 10

Microarrays

Answer 10

Microarrays are a technique used to measure thousands of transcripts for multiple samples in one go.

Question 11

Microarrays: Affymetrix

Answer 11

1. Start with Total RNA : The process begins with the isolation of Total RNA from a sample. The diagram shows mRNA (a component of Total RNA) with a poly-A tail (AAAA).
2. Reverse Transcription to cDNA: The RNA is converted into complementary DNA (cDNA) using the enzyme reverse transcriptase.
3. In Vitro Transcription (IVT) to Biotin-labeled cRNA: The cDNA is then used as a template to synthesize complementary RNA (cRNA) in a process called In Vitro Transcription. This cRNA is labeled with biotin(indicated by the ‘B’ next to the cRNA).
4. Fragmentation: The long biotin-labeled cRNA molecules are broken down into Fragmented, Biotin-labeled cRNA.
5. Hybridization: The fragmented, biotin-labeled cRNA is incubated with the GeneChip Expression Array to allow hybridization. The cRNA (the target) binds to its complementary sequence (the probe) that is already synthesized on the chip surface.
6. Wash and Stain: The array is washed to remove non-specifically bound targets. A fluorescent stain (which binds to the biotin label ‘B’) is applied to label the hybridized fragments.
7. Scan and Quantitate: The array is placed into a scanner. The scanner detects the fluorescent signal (light intensity) at each probe, which is proportional to the amount of hybridized cRNA. This raw data is then analyzed and quantitated to determine the level of gene expression.

Question 12

Microarrays: spotted vs oligonucleotide

Answer 12

Spotted microarrays:
* cDNA, oligos, PCR fragments
* Synthesized prior to deposition on a chip or slide
Oligo microarrays:
* short fragments (~25 to 60 nt)
* Synthesized directly on the array surface (photolithography)

Question 13

Microarrays: 2-channel vs 1-channel

Answer 13

2-Channel Microarrays
* 2 sample sets are labeled with different dyes
* Measures relative differences, not absolute amounts
* Results difficult to compare
Single-Channel (“one-color”)
* 1 chip per sample
* Easier to compare different experiments
* Popular systems:
-> Affymetrix
-> Illumina
->Agilent

Question 14

2-Channel Microarrays

Answer 14

• Two sample sets are compared on a single chip.
• Each sample is labeled with a different fluorescent dye (e.g., “Red Fluorescent” probes for one sample and “Green Fluorescent” probes for the other).
• The labeled samples are combined and hybridized to the microarray.
• The results measure relative differences in expression, not absolute amounts.
• Results can be difficult to compare between different experiments.
• Example of results: Spots on the array can be Red (present in pathological cells only), Green (present in normal cells only), Yellow (present in both cells), or Gray/Black (not present in cells).

Question 15

Single-Channel Microarrays (“One-Color”)

Answer 15

• One chip is used per sample.
• They are easier to compare between different experiments.
• Popular systems include Affymetrix, Illumina, and Agilent.
• The Affymetrix process involves converting Total RNA to cDNA, then to Biotin-labeled cRNA via In VitroTranscription, followed by fragmentation, hybridization, wash and stain, and finally scan and quantitate.

Question 16

Explanation of the Data Table - Table Elements

Answer 16

• ID_REF: This column contains the unique identifiers for the probes on the microarray. Each identifier (e.g., 10a10b13c) corresponds to a specific gene or sequence being measured.
• GSM155812 through GSM155816: These column headers represent the different samples (or experimental conditions/replicates) that were analyzed on the microarray. The ‘GSM’ prefix suggests the data was retrieved from the Gene Expression Omnibus (GEO) public database.
• NA (Not Available): This indicates that there was no reliable measurement for that specific probe in that specific sample (e.g., the signal was too low, or the data point was filtered out during quality control).

Question 17

Explanation of the Data Table - Interpreting the Numerical Values (Log Ratios)

Answer 17

The values in the table are typically the log2​ ratio of the relative gene expression, which measures the relative difference in gene expression, not the absolute amount.

Question 18

Explanation of the Data Table -Interpreting the Numerical Values

Answer 18

Question 19

Mining Public Microarray Data

Answer 19

Data from microarray experiments is often deposited in public databases, which can be mined for analysis.
* GEO (Gene Expression Omnibus, NCBI)
* ArrayExpress (EBI)

Question 20

RNA-seq

Answer 20

• RNA sequencing is mentioned as another technique for studying gene expression.
• It typically analyzes fragments of 50−300bp.

Question 21

RNA-seq - In vivo: RNA Generation

Answer 21

• DNA gene in genome: A gene sequence on the DNA serves as the template.
• Transcription: The DNA is transcribed into a Pre-mRNA molecule.
• Intron splicing: Non-coding regions (introns) are removed, and coding regions (exons) are joined together to form the Mature mRNA. The Mature mRNA is the functional transcript that is typically quantified in RNA-seq.

Question 22

RNA-seq - In vitro: Library Preparation and Sequencing

Answer 22

• FRAGMENTATION: The isolated RNA (primarily Mature mRNA) is broken down into small RNA fragments.
• REVERSE TRANSCRIPTION: The RNA fragments are converted into double-stranded complementary DNA (ds-cDNA fragments). cDNA is more stable and is required for most sequencing platforms.
• HIGH-THROUGHPUT SEQUENCING: The ds-cDNA fragments are sequenced to produce millions of short pieces of sequence data called Short-read sequences (typically 50−300 bp long).

Question 23

RNA-seq: Computational Analysis (In silico)

Answer 23

• Align to genome: The Short-read sequences (RNA-Seq reads) are mapped and aligned back to the known Genome sequence.
• QUANTIFY EXPRESSION: The expression level is quantified by counting the number of reads that align to a specific gene.
• Results: The output is usually presented as a normalized count, such as “counts per gene per million bp.”

* Gene A has a high count (e.g., 50,000 reads), indicating high expression.
* Gene B has a low count (e.g., 10,000 reads), indicating lower expression.

• Sequence processing and Alignment: The short-read sequences are aligned to the Genome sequence, often spanning exon-junctions.
• Identifying Splice Variants: By observing how the reads align across the genome—specifically where the reads “jump” over large genomic regions—researchers can infer how the pre-mRNA was spliced. This allows for the identification of different splice variants (or isoforms) of the same gene.

* Splice variant A might include a particular exon.
* Splice variant B might exclude that exon, showing an alternative form of the mature mRNA.

Question 24

ChIP-seq

Answer 24

ChIP-seq addresses several key biological questions:
* Binding Location: Where in the genome does a specific protein bind?
* Target Genes: What are the target genes of a particular transcription factor (TF)?
* Binding Sequence: What is the specific DNA sequence that the protein binds to?

Question 25

ChIP-seq - Method

Answer 25

1. **Sample Fragmentation and Immunoprecipitation:** Non-histone ChIP: A specific antibody targets the non-histone protein bound to the DNA. 2. **Histone ChIP**: An antibody targets a specific modification on a histone protein. The antibody pulls down (immunoprecipitates) the protein along with the DNA fragment it's bound to. 3. **DNA Purification**: The DNA fragments are separated from the proteins. 4. **Sequencing**: The purified DNA fragments are sequenced using Next-Generation Sequencing (NGS) technology.

Question 26

ChIP-seq data formats

Answer 26

The raw sequencing data is processed through a bioinformatics pipeline: 1. **RAW Data**: The initial data consists of sequence reads (short DNA sequences). These are often stored in archives like the "Small Read Archive (SRA)". 2. **Alignment to Genome**: The sequence reads are mapped (aligned) back to the reference genome. 3. **Peak Calling**: Regions in the genome with a significantly high density of aligned reads (i.e., high "read coverage") compared to a background are identified as "ChIP peaks". These peaks indicate the protein's binding sites.

Question 27

ChIP-seq: “Sequence logo”

Answer 27

Sequence Logo: The actual DNA binding motif (the sequence the protein prefers to bind to) can be visualized using a "Sequence logo".

Question 28

Finding public ChIP-seq data

Answer 28

Publicly available ChIP-seq data can be found in several databases: * NCBI Gene Expression Omnibus (GEO) * EMBL European Nucleotide Archive (ENA) * ENCODE Project (Note: All ENCODE data is also available on GEO)

Question 29

Next-Generation Sequencing

Answer 29

-> NGS is the technology used to read the DNA fragments from ChIP-seq (and RNA-seq) experiments. **Common NGS Platforms** Several platforms are used for NGS: * Illumina (Solexa) sequencing (Currently most used for RNA-seq and ChIP-seq) * Roche 454 sequencing * Ion Torrent: Proton / PGM sequencing * Nanopore sequencing

Question 30

Illumina Sequencing Process

Answer 30

**Library Preparation:** * The sample DNA is fragmented. * Adaptors (short, synthetic DNA sequences) are added to the ends of the DNA fragments. **Flow Cell Attachment and Amplification:** * The adaptor-ligated fragments attach to a glass slide called a flowcell. * Bridge amplification & cluster formation is performed, which copies each fragment multiple times, creating localized clusters of identical DNA molecules. **Sequencing-by-Synthesis (SBS):** * The DNA strands are sequenced one base at a time. * Reversible, fluorescently labeled terminators (one for A, T, C, G) are added to the flowcell. * At each cycle, one labeled base is incorporated into the growing strand. * A camera images the flowcell to record the color (and therefore the base) incorporated at each cluster site. * The fluorescent label and the terminator are chemically cleaved, allowing the next cycle to begin. **Sequence Construction:** * Computers detect the base at each site in the images from all cycles. * These sequential base detections are used to construct the final sequence (the "read"). * All sequence reads produced are the same length, determined by the number of cycles performed.