BioTech Notes

Question 1

Flo Cytometry Outline

Answer 1

qualitive and quantitive cell analysis (large data processing). Measures biological and physical properties of cells (cell count, phenotype + health). Laser shines on single cell (in saline) at a time (forward (size, x-axis)+ side scatter (y-axis))

Question 2

Flo Cytometry Principles

Answer 2

Label cells suspensions with antibodies against different targets. Antibodies are conjugated to different flurophores (eg Propidium iodide = red (chromosomes), annexin V = green (phosphatidylserine membrane)

Question 3

Flow Cytometry Applications

Answer 3

Protein expression, Translational modifications, cell health/cycle status, cell sorting and multiplex bead arrays

Question 4

Immunophenotyping in Flow Cytometry Example

Answer 4

Detection of murine regulatory T cells (CD4, CD25 and FOX3)

Question 5

Fluorescence Activated Cell Sorter Method

Answer 5

Fluroescent antibodies bind to cells, samples are loaded into machine, Laser beam strikes each drop (FSC = size, SCC = complexity), electrode assigns droplet + or - charge, parallel oppositely charged plates attract drops to different buckets (separated based on charge)

Question 6

Fluroescent Activated Cell Sorter Applications

Answer 6

Cancer research (isolate + identify cancer cells from samples, diagnosis), immunology research (separates cells based on surface markers), stem cell research and developmental biology research (different cells for different stages of embryo dev)

Question 7

Function of Multiplex Bead Arrays

Answer 7

Generates standard curves to calculate cytokine concentrations

Question 8

Reverse Transcription PCR Method

Answer 8

RNA reverse transcribed into complementary DNA using reverse transcriptase. cDNA is amplified by PCR. Allows further study of original RNA and mRNA

Question 9

Reverse Transcription PCR Application

Answer 9

Gene expression detection, examine transcript variants and generate cDNA for cloning/dequencing

Question 10

How to perform quantitive PCR after RT-PCR

Answer 10

cDNA is denatured. Random primers (may contain fluroescent tabs) are added to cDNA in eppindorfs (annealing) and mixed in thermocycler (elongation). Then ran through qRT-PCR machine

Question 11

Quantative RT-PCR

Answer 11

RNA is converted by reverse transcriptase into cDNA. cDNA is amplified by PCR (denaturation, annealing and elongation)

Question 12

Flurophores in real time PCR fluroescence

Answer 12

Flurophore in ground state; denaturation and primer annealing. Flurophore in excited state; dsDNA and primer extension

Question 13

Applications of qRT-PCR

Answer 13

Infectous disease id (from nucleic acid: Hep B, covid, HIV), cancer abnormalities (known transcripts), research (cell differntiation in tissue after drug exposure), microbiology (food safet/spoilage, microbrial risk)