Transcriptomics Flashcards
(16 cards)
what is the purpose of RNA-seq (bulk RNA sequencing)?
- quantifies gene expression across a population of cells
- captures the transcriptome at a given time
steps in RNA-seq & key reagents
- RNA isolation: total or mRNA extraction using kits (e.g., with oligo-dT beads to pull poly-A mRNA).
- cDNA synthesis: reverse transcription using reverse transcriptase (converts RNA → cDNA).
- Library prep: fragmentation of cDNA, adapter ligation (for flow cell attachment), indexing for multiplexing.
- Sequencing: Illumina NGS typically—sequencing by synthesis.
- Data analysis: align reads to a reference genome, quantify transcript levels.
key reagents:
- reverse transcriptase = for cDNA synthesis
- adapter sequences = for flow cell ligation
= RNA purification reagents = for RNA isolation
what does scRNA-seq do? (Single-Cell RNA Sequencing)
profiles transcriptomes at single-cell resolution—detects cellular heterogeneity
what do microarrays do?
analyse the expression levels of multiple genes simultaneously - snapshot of the transcriptome (all the RNA transcripts) within a cell or sample
steps in conducting a microarray?
- isolate RNA → convert to cDNA (reverse transcriptase) & label with fluorescent dyes (e.g. Cy3/Cy5).
- hybridize to DNA probes on a chip (each probe complementary to a known gene).
- scan for fluorescence intensity → reflects expression level.
what does NGS do? (next generation sequencing)
high-throughput sequencing of DNA/RNA - determines the sequence
what are the 4 main steps in NGS?
library preparation
cluster generation
sequencing by synthesis
data analysis
outline the 4 steps of NGS & what happens
- library preparation
- DNA sample is fragmented & adapter sequences ligated to both ends
- adapters allow attachment to the flow cell and serve as primer-binding sites for sequencing - cluster generation
- adapter-tagged DNA fragments bind to complementary oligonucleotides on the flow cell surface (oligos complementary to the adapter sequences)
- bridge amplification = bound DNA bends to form a bridge, hybridizing to a nearby oligo
- DNA amplifies this fragment - repeated cycles form clonal clusters, enhancing signal strength - sequence by synthesis
- primers anneal to adapters flanking each DNA cluster
- DNA polymerase adds one fluorescently labelled, reversibly terminated nucleotide per cycle
- after each cycle - laser excites fluorophore = imager captures signal (colour = base) = fluorophore and terminator cleaved = cycle starts again - data analysis
- fluorescence signals are converted into sequence reads; reads are aligned to a reference genome
what does qPCR do?
uses fluorescent dyes or probes to detect and measure the amount of DNA as it’s being amplified
steps in qPCR & key reagents
- cDNA synthesis from RNA (reverse transcriptase)
- amplification of gene-specific regions using fluorescent dye (SYBR Green) or probe-based detection (TaqMan)
- amplification requires DNA polymerase - measure fluorescence intensity after each cycle → quantifies gene expression in real-time
Which of the following steps is not required before sequencing in RNA-seq?
A. RNA fragmentation
B. Reverse transcription to cDNA
C. Adapter ligation
D. DNA denaturation
D. DNA denaturation
What is the main advantage of single-cell RNA-seq over bulk RNA-seq?
A. Higher throughput
B. Lower sequencing cost
C. Captures cell-to-cell variation in gene expression
D. Avoids the need for reverse transcription
C. Captures cell-to-cell variation in gene expression
Microarray technology differs from RNA-seq because:
A. It doesn’t require RNA isolation
B. It sequences RNA directly
C. It uses known probes to detect transcripts
D. It can detect novel transcripts
C. It uses known probes to detect transcripts
Which of the following best explains why qPCR is used after RNA-seq?
A. To visualize protein localization
B. To confirm sequencing depth
C. To validate expression changes of specific genes
D. To replace the need for sequencing altogether
C. To validate expression changes of specific genes
List and briefly describe the 4 main steps of Illumina sequencing by synthesis (SBS).
- DNA cluster already generated through bridge amplification & DNA polymerase activity
- fluorescently-labelled dNTPs/bases that can be reversibly terminated are added - one base per cycle
- once a base is added, fluorophore in base is excited, emits a fluorescence due to Stokes Shift - each fluorescence = a colour unique to a base
- imaging software records the colour & analyses it - constructs a sequence reading
- base is then cleaved & cycle starts again
describe the entire workflow of an NGS Illumina sequencing protocol
- Library Preparation
DNA is fragmented and ligated with adapter sequences that allow it to bind to the flow cell and serve as priming sites. - Cluster Generation
Fragments hybridize to oligos on the flow cell and undergo bridge amplification, forming dense clusters of identical sequences for high signal detection. - Sequencing by Synthesis
Reversible terminator nucleotides with fluorescent tags are incorporated one at a time. A laser excites each base, emitting a unique signal detected by imaging. The tag is cleaved, and the next cycle begins. - Data Analysis
Fluorescence data is processed to determine the base sequence at each cycle. Reads are aligned to a reference genome or transcriptome for further analysis.
