Transcriptomics Flashcards
How do we measure how much a gene is expressed?
What is PCR
How is qPCR done with a reporter probe
How does qPCR work
What is qPCR normalisation
Use the total amount of RNA or DNA in each sample as a normalization factor.
Quantify RNA/DNA input before reverse transcription or amplification and adjust qPCR data accordingly.
What are the limitations of qPCR
What are microarrays
How does an Affy Gene chip work*
An Affymetrix GeneChip (or Affy GeneChip) is a microarray technology used to measure gene expression or detect variations in DNA, such as SNPs
A glass or silicon chip is embedded with thousands to millions of unique oligonucleotide probes.
Each probe is complementary to a specific sequence of the target RNA or DNA.
Perfect Match (PM) Probes: Complementary to the target sequence.
Mismatch (MM) Probes: Have a single base mismatch to account for nonspecific hybridization.
Total RNA or mRNA is extracted from the sample and reverse transcibed into cDNA
e labeled cRNA is fragmented to ensure optimal binding and hybridized onto the chip.
The cRNA binds to complementary probe sequences on the array through base-pairing rules.
The chip is scanned using a laser scanner to measure fluorescence intensity at each probe location.
Fluorescence Intensity: Proportional to the amount of hybridized RNA, which reflects the expression level of the corresponding gene.
What are the limitations of using a microarray
How does next generation sequencing work
What can RNA -seq be used to do
Give an overview of rna-seq
How is rRNA removed in RNA-seq
How is RNA prepared for sequencing
What is RNA-seq normalisation and how is it done
What is single cell RNA-seq + problems with RNA-seq
What is the Single Cell RNA-seq Workflow
How is Single Cell RNA-seq Data Analysed*
Build a graph of cell-cell similarities (e.g., k-nearest neighbors) and identify clusters
How can data from Single Cell RNA-seq be plotted
What methods can be used for profiling binding of transcription factors and histone modifications
What can CHIP sequencing be used to do
Give the Chip-seq Workflow
How is the data visualised from Chip-seq analysis*
Peaks representing regions of DNA enriched for the protein of interest are seen
What are the limitations of Chip-seq
How does cut and run work*
The chromatin is maintained in its native state without crosslinking
A specific antibody targeting the protein of interest (or histone modification) is added.
The antibody binds to its target protein on the chromatin.
A fusion of protein A or protein G (which binds to the antibody) and micrococcal nuclease (MNase) is introduced.
The MNase is tethered to the chromatin via the antibody-protein interaction
The nuclease selectively cleaves DNA near the bound protein-antibody complex.
Cleaved DNA fragments are released into the supernatant, while other chromatin remains intact.
The released DNA fragments are purified.
These fragments correspond to regions of the genome where the protein of interest was bound
The isolated DNA is prepared for sequencing
