Transcriptomic approaches & working with proteins

Question 1

What is bioinformatics?

Answer 1

field of study that combines biology and computer science to analyse and interpret large sets of biological data such as DNA and protein sequences, gene expression data, and protein structures.

Question 2

What can we use bioinformatics for?

Answer 2

Big data analysis:
-protein structure prediction
-sequence alignment
-phylogenetics
-homology modelling

Question 3

How is a DNA library prepared?

Answer 3

1. extraction of RNA from a sample e.g blood, saliva, tissue
2. RNA convert to DNA
3. Shear dsDNA fragments
4. attach adapters to fragments
5. library formation

Question 4

How is the DNA library sequenced?

Answer 4

1. deposited into flowcell
2. bridge amplification to form clusters
3. Flowcell loaded on machine
4. sequencing by synthesis
5. Annealing of sequencing primer
6. Sequence each nucleotide 1 cycle at a time in a controlled manner
7. Modified 4 bases (ATCG) with reversible terminators AND a different fluorescent dye tag
8. Single nucleotide incorporation (DNA polymerase)
   9.Flowcell wash
9. Image the 4 bases (digital photograph)
10. Cleave chain terminator chemical group and dye with enzyme (repeat steps 8-11 for full length sequence)
11. Camera sequentially images all 4 bases on the surface of the flowcell each cycle. Each cycle image is converted to a nucleotide base call (A or C or G or T). Cycle number anywhere between 50 – 250 nucleotide base pairs, depending on desired sequence length

Question 5

What are the main steps of DNA library sequencing?

Answer 5

1.DNA library preparation
2.Library amplification via PCR
3.Library sequencing: The DNA library is sequenced using one of several sequencing methods, such as Sanger sequencing, Illumina sequencing, or nanopore sequencing.
4.Data analysis

Question 6

What is the transcriptome?

Answer 6

The transcriptome refers to the complete set of all RNA molecules produced by a cell, tissue, or organism. An important tool for understanding gene expression and regulation. RNA seq a common technique used to study it

Question 7

What is gene expression?

Answer 7

Gene expression refers to the process by which the information encoded in a gene is used to synthesize a functional gene product, such as a protein or a non-coding RNA molecule.

Question 8

What do RNA seq experiments use?

Answer 8

use the total RNA (or mRNA) from a
collection of cells or tissue

Question 9

What is RNA seq?

Answer 9

the study of gene expression at a molecular level. It involves the sequencing and analysis of the RNA molecules present in a biological sample, such as a tissue or cell.

Question 10

How does RNA seq work?

Answer 10

1. converted to cDNA prior to library construction
2. NGS to determine which genes are actively expressed
3. a single experiment can capture expression levels of thousands of genes
4. number of sequencing reads produced from each gene can be used as a measure of gene abundance
5. Quantification of the expression levels
6. With appropriate analysis, RNA-seq can be used to discover distinct isoforms of genes are differentially regulated and
   expressed

Question 11

What is a standard fastq file?

Answer 11

1) Sequence ID
2) Nucleotide Sequence
3) Strand
4) Per base quality score

Question 12

What is alignment in RNAseq data analysis?

Answer 12

1. Align short sequence reads (the
   fastq files) to the reference
   genome
2. Specialist bioinformatic
   alignment programs
3. Alignment file

Question 13

How is RNAseq data typically presented?

Answer 13

A volcano plot is a typical way to present
RNAseq results. Plot of log2 fold changes versus P-value for significance (-log10). Shows the genes that are up (green) and down regulated.

Question 14

What is Gene Set Enrichment Analysis (GSEA)?

Answer 14

is a computational method that determines whether a priori defined set of genes shows statistically significant, concordant differences between two biological states. uses a priori
gene sets that have been grouped together by their involvement in the same
biological pathway. the fc
focus is put on a gene set.

Question 15

What is differential gene expression?

Answer 15

Differential gene expression refers to the differences in the expression levels of genes between different samples or conditions. It occurs when the amount or activity of a particular gene is different between two or more groups being compared, such as between a healthy and diseased tissue, or between a treatment group and a control group.

Question 16

what is a Bradford assay?

Answer 16

laboratory method used to quantify the concentration of proteins in a sample. The assay is based on the binding of Coomassie Brilliant Blue dye to the amino acid residues in the protein, resulting in a shift in the dye’s absorbance spectrum and a change in color from brown to blue.

Question 17

How is the colour change measured in a Bradford assay?

Answer 17

a spectrophotometer. The amount of protein in the sample can be determined by comparing the absorbance of the sample to a standard curve generated using known concentrations of a protein of interest.

Question 18

What is SDS-PAGE?

Answer 18

SDS-PAGE stands for Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. It is a laboratory technique used to separate proteins based on their size and charge.

Question 19

How does SDS-PAGE work?

Answer 19

a protein sample is mixed with a detergent called sodium dodecyl sulfate (SDS), which denatures the proteins and coats them with a negative charge. The sample is then loaded onto a polyacrylamide gel, which is composed of a cross-linked polymer matrix.

When an electrical current is applied, the negatively charged SDS-coated proteins migrate towards the positively charged anode, and their movement through the gel is impeded by the matrix, which acts as a molecular sieve. Smaller proteins move more easily through the matrix and thus migrate farther through the gel than larger proteins.

After the electrophoresis is complete, the gel can be stained with a protein-specific dye, such as Coomassie Brilliant Blue or silver stain, to visualize the separated proteins. The separated proteins can then be cut from the gel for further analysis, such as protein identification by mass spectrometry.

Question 20

What is western blotting?

Answer 20

also known as immunoblotting, is a laboratory technique used to detect and identify specific proteins in a sample. It is based on the principle of separating proteins by SDS-PAGE and then transferring them to a membrane, where they can be detected using protein-specific antibodies.

Question 21

How does western blotting work?

Answer 21

a protein sample is first separated by SDS-PAGE and transferred to a membrane, usually made of nitrocellulose or PVDF. The membrane is then incubated with a primary antibody that is specific to the protein of interest. The primary antibody binds to the protein and is then detected using a secondary antibody that is conjugated to an enzyme or a fluorescent dye. The secondary antibody binds to the primary antibody and generates a signal that can be visualized using techniques such as chemiluminescence or fluorescence.

Question 22

How to prepare a whole cell extract? (cell lysate preparation)

Answer 22

1. Collect the cells: Grow the cells of interest in culture and harvest them using a suitable method, such as scraping or trypsinization.
2. Wash the cells: Centrifuge the cells and wash them with a buffer solution, such as phosphate-buffered saline (PBS), to remove any residual media or serum.
3. Resuspend the cells: Resuspend the cells in a lysis buffer, which typically contains detergents, salts, and protease inhibitors, to disrupt the cell membrane and release the cellular components.
4. Lyse the cells: Lyse the cells by subjecting them to mechanical disruption, such as sonication or homogenization, or by repeated freeze-thaw cycles.
5. Centrifuge the lysate: Centrifuge the lysate to remove the insoluble cellular debris, such as nuclei and cell membranes, and collect the supernatant, which contains the soluble proteins and other cellular components.
6. Quantify the protein concentration: Measure the protein concentration of the extract using a suitable method, such as a Bradford assay or a BCA assay.

Question 23

How is a standard curve used in a Bradford assay?

Answer 23

used to estimate the protein concentration of an unknown sample based on its absorbance signal. The standard curve is typically generated by measuring the absorbance of a series of known protein standards with known concentrations.

Question 24

what are the general steps of a Bradford assay?

Answer 24

1. Prepare a series of protein standards with known concentrations
2. Prepare the protein samples of interest and dilute them to an appropriate concentration with a buffer solution.
3. Add a small amount of Bradford reagent, which contains Coomassie Brilliant Blue dye, to each standard and sample.
4. Incubate the standards and samples for a specified time, typically around 10 minutes, to allow the color to develop.
5. Measure the absorbance of each standard and sample at a specific wavelength, such as 595 nm, using a spectrophotometer.
6. Use the absorbance readings to calculate the protein concentration of the samples using the standard curve generated from the known protein standards.

Question 25

How to make an SDS-PAGE gel?

Answer 25

1. Prepare the separating gel
2. Place a gel cassette in the gel apparatus and insert a comb to create wells for loading samples.
3. Pour the gel mixture into the cassette, avoiding air bubbles. Allow the gel to polymerize by incubating it for around 30-60 minutes.
4. Prepare the stacking gel
5. Pour the stacking gel mixture on top of the separating gel, being careful not to disturb the separating gel. Place a comb in the stacking gel to create wells.
6. Allow the gel to polymerize by incubating it for around 30-60 minutes.
7. Remove the comb
8. Rinse the wells with running buffer to remove any unpolymerized acrylamide.

Question 26

How to run an SDS-PAGE gel?

Answer 26

1. Add protein samples, mixed with a loading buffer, to the wells in the SDS-PAGE gel.
2. Connect to a power supply
3. Run the gel
4. Once the proteins have migrated to their desired positions, turn off the power supply and remove the gel cassette from the apparatus.
5. stain the gel to visualise separated proteins
6. destain to remove excess stain to enhance visibility of protein bands
7. image gel