What is the purpose of transcriptome studies?
- Understanding how a gene is regulated (expressed) gives important information about its potential role
- characterization of non-coding RNA
- quantification of gene, ability to quantify the level at which particular gene is expressed within a cell, tissue or organism
- there are diffrent methods to study gene expression ( PCR-based methods, Microarray hybridisation and RNA-seq)
Gene expression quantification:
PCR-based methods
semi-quantitative (not quantitative) PCR
- Semi-quantitative PCR relies on the amplification of the target DNA through the polymerase chain reaction.
- The intensity of the resulting PCR product is used as an indirect measure of the initial amount of the target DNA in the sample.
- dsDNA
- denaturation
- primer anneling
- extension
(each cycle doubles the DNA)
quantitative PCR (qPCR)
- Detection and quantitation of PCR amplicons throughout a reaction/after
each cycle (thus, “real-time”)
- You can “see” and measure how much PCR product is produced after each cycle
- depending on detection chemistry e.g: via accumulation and measurement of specific fluorescent sigbnals with each cycle (SYBR Green I)
- Use of a fluorometer to measure the amount of fluorescent in every cycle
On DNA: qPCR (Quantitavie PCR)
On RNA: RT-qPCR (Reverse transcription quantitave PCR) . The reverse transcriptiton step is to convert RNA to complementary DNA (cDNA) before the PCR amplification
Method:
There are 4 phases during the amplification PCR
1. Linear-ground phase
2. Exponential phase
3. Long-linear phase
4. Plateau phase
During phase 2 and 3:
* PCR product begins to amplify
* High efficiency
* Doubling of product at each cycle
During Phase 4:
* Primer depletion
* Degradation of reagents
* no prodct accumulation
What is the role of SYBR green in quantitative PCR?
SYBR green I is a fluorescent dye that bind to dsDNA
* resulting DNAdye-complec sborbs blue light and emits green light
* As DNA is amolifies, SYBR GReen fluorescent increases proportionally
* The amount of fluorescence is proportional to the (log) amount of dsDNA.
How does RT-qPCR work?
RT-qPCR = qPCR of RNA = quantitative-reverse transcription PCR
- RNA is converted to DNA (mRNA to cDNA) = reverse transcription
- The more copies of target/cDNA there are at the beginning of the assay, the fewer cycles of amplification are required for the fluorescence to reach the threshold level of detection
PCR reaction:
* cDNA (template)
* Primers
* qPCR master mix (polymerase, PCR buffer, nucleotides, SYBR Green I)
- During the PCR cycling the fluorescence is measured in “real-time”
- High-troughout (In less then 2 hours 284 amplification plots)
How is signal intensity determined in RT-qPCR?
Signal intensity is determined by monitoring the fluorescence emitted during the amplification process. (SYBR Green I)
Quantification:
* The Ct value represents the cycle number at which the fluorescence signal reaches a specified threshold.
* A lower Ct value corresponds to higher initial target RNA/DNA concentration in the sample.
Why RT-qPCR?
Advantages:
– Low detection limit (< 10 molecules can be measured)
– High precision (repeatable measures)
– Low costs for low number of transcripts (typically < 0.5 €)
Drawbacks:
– Depends heavily on enzymes, and quality of upstream sample handling
– High costs for high number of transcripts
Challenges:
– Proper primer design
– Measurement (handling) and analysis of large number of transcripts
– Normalization of data
Genome-wide transcriptome analysis:
microarrays
Microarry-based methods
* a microarry is a tool for analyzing gene expression that consists of a small memebrane or glass slide containing samples of many gene arranged in regular pattern
- Grid containing DNA probes for many or even all of the genes of an organism
–> Many copies of a single stranded DNA probes tethered to a solid support - RNA is extracted from the sample of interest (e.g: cells or tissue)
- RNA is reverse transcribed into cDNA and labeld with fluorescent syes
- labeled cDNA is applied to the microarray
- Labeld cDNA bind specifically to its complementary probe (washing etc..)
- Microarray is analyzed in a scanner, which uses laser light to cause each sport to fluoresce and measure the relativie intesity of emission, typically green and red wave lengths
–> green spot: gene is expressed in tissue 1
–> red spot: gene is expressed in tissue 2
–> yellow spot: gene is expressed in both tissued
–> no coloring: gene is not expressed in either tissue
Genome-wide transcriptome analysis:
Affymetrix
- Affymetrix was a biotechnology company developed GeneChip microarrays, allowing researchers to analyze the expression of thousands of genes simultaneously.
- Affymetrix microarrays were used for gene expression profiling, SNP genotyping, and comparative genomic hybridization (CGH).
- The technology used short DNA sequences called probes immobilized on a solid surface.
- Each probe 25 nucleotides long
- several probe pair per gene
- Mismatch pair: single mismatch at centre of probe
- Used to detect & eliminate false results
- Direct synthesis of oligonucleotides (probes) on the glass surface using
photolithography
While Affymetrix microarrays were widely used, next-generation sequencing (NGS) has become more popular for comprehensive genomic studies
RNA-seq principle and workflow
RNA-seq is a high-throuput sequencing technique used to analyze ant quantify the transcriptome, providing a comprehensive view of the RNA molecules
Benefit of RNA-seq: this is an unbiased approach; means that also the abundance of transcripts can be measured that were not known before
(difference to qRT-PCR and microarrays)!
Workflow:
1. Isolate RNA
2. Break the RNA into small fragments ( We do this because RNA transcripts can be thousand of bases long, but the sequencing machine can only sequence short fragments (200-300 bp)
3. Convert RNA fragments into double stranded cDNA. dsDNA is more stable than RNA and can be easily amplified and modiefied
4. Add sequencing adaptors
5. PCR amplify
6. Sequrence cDNA using sequencing platforms. Requieres NGS technologies e.g Illumina Sequencing
7. Analysis (bioinformatic)
The number of reads aligned to each gene or transcript is used as a measure of its abundance. This quantification allows for the estimation of gene expression levels
Principal of Laser capture microdissection (LCM)
- LCM is a method to procure subpopulations of tissue cells under direct microscopic visualization
- harvest the cells of interest directly
- can isolate specific cells by cutting away unwanted cells - used to discover which genes are expressed in these cells (cut-out tissue)
Varied downstream applications are possible:
* Transcriptome analysis (RNA-seq)
* DNA genotyping
* Proteomic studies
Disadvantges:
* relativly low throughput
* technically challenging
Methods:
- UV-LCM (with laser beam)
- IR-LCM (with plastic film)
Single-cell RNA-seq (scRNA-seq) verus Bulk-seq
scRNA-seq: (Fruit bowl)
the expression data are repressented at the individual cell level, Poroviding information on how diffrent cell types impact overall expression.
Provides deeper insight to the multi-tiered complexity of different cells within the same tissue type.
1. Isolated cells are lysed
2. mRNA molecules, which conatin poly(A) tail, are captured cia the polyT-primers on the beads. CApturing rRNA is largely avoided
3. RT-transcriptase converts the PolyT-primed mRNAs into cDNA
4. PCR amplifies the cDNA molecules. Sometimes, in vitro transcription, followed by another round of reverse transcription, is employed.
5. Barcoding tags and the other short sequences required by the sequencing platform are added to cDNA molecules
Bulk-seq: (Smoothie)
Output data are averaged expression data compared across sample or othe rtypes of comptational analyses applicable to bulk data
Single-cell RNA-seq (scRNA-seq) using combinatorial indexing (barcoding)
Split-pool ligation-based transcriptome sequencing (SPLiT-Seq) . A scRNA-seq method that labels the cellular origin of RNA through combinatorial barcoding.
SPLiT-Seq ist compatible with fixed cells or nuclei
1. Reverse Transcription
Split. samples are disturbed into wells and the first sample-specific barcodes are applied to fixed cells or nuclei with an in-cell rverse trancription (RT) reaction
2. Pool.
Cells from each well are pooled together
3. Ligation.
Split. Cells or nuclei are disturbed across a plate and an in-cell ligation append the second barcode
4. Pool.
Cells from each well are pooled together
5.Ligation
Split.A third barcode is applied with another in.cell ligation after the cells or nuclei are split across plate
6.Pool
Cells from each well are pooled together
7.Lysis+PCR
Split. The pooled cells are divided across veral sublibaries. The cells are lysed and the fourth, sublibary specifc barcode is applied by PCR
8.Library Prep
Split. The library prperation appends adapters ready for loading on any illumina sequencer
Pros and Cons of scRNA-seq
Pros:
* No need to generate transgenic organisms
* No a prior information about cell types needed
* New cell types can be discovered
Cons:
* Still very expensive (2,000 EUR per sample)
* Weakly expressed genes are difficult to detect
* It is not necessarily known from which cell type the mRNA originated
Ribosome profiling, bulk approach
Ribosome profiling = Ribo-seq = ribosome footprint
- a technique to study genome-wide mRNA translatiob through deep sequencing (via NGS) of ribosome footprints
- It maps and quantifies ribosome occupany on mRNAs, which enables the identification of coding regions and the accurate quantification of translatiob efficiency
- It allows to determine which mRNAs are being actively translated
The bulk approach: (biochemical)
1. Inactivate ribosome-mRNA complexes by treatment with cycloheximide (an inhibitor of translation)
2. Lyse cells or tissues and isolate ribosomes with the mRNA molecules bount to them.
3. Isolate the mRNA-ribosome complexes by sucrose gradient density centrifugation (or specialized chromatography columns).
4. Treat with ribonuclease to degrade mRNA segments not covered by ribosomes.
5. Isolate mRNA segments previously protected (by ribosomes) from degradation.
6. Sequence those protected mRNA segments by NGS (e.g., Illumina sequencing).
7. Align sequence results to the genomic sequence to determine the translational
profile.
there is also a method to isolarte ribosomes and their attached mRNAs, in a cell-specifc manner –> cell-specific approach
