RT PCR

Goal of rtPCR

Detect presence of a target gene

Two dyes using intercalating method to detect PCR product

1. SYBR green

2. EtBr

On which ends are the fluorescent reporter and quencher on TaqMan

Fluorescent reporter is on 5’ end

Quencher on 3’ end

How does the DNAP remove the TaqMan probe

via 5’→3’ endonuclease activity

Draw the rtPCR graph

What should be done to properly calculate PCR Efficiency

Make serial 10-fold dilutions with known quantities of starting sample and plot them on a graph to get standard curve.

How to determine reaction specificity

Use a melt curve.

1. SYBR binds to dsDNA

2. Increase the temperature

3. As the temperature decreases, strands denature, resulting in lower fluorescence

2 purposes of absolute gene quantification

1. Check for chromosome or gene copy number

2. Measure viral loads

Goals of absolute and relative quantifications

1. Absolute focuses on gene or chromosome number, and viral loads

2. Relative focuses on tracking gene expression

Role of CYP1A

Detoxification of toxic compounds such as polyaromatic hydrocarbons (PAH)

What properties of cells can flow cytometry measure

1. Size

2. Granularity

3. Fluorescence

3 pieces of flow cytometry

1. Fluidics, separate cells into droplets

2. Optical system

3. Electronic system

What principle does flow cytometry rely on to put cells individually into each droplet

Hydrodynamic focusing, involves injecting sample into sheath fluid which separate it into individual drops

FSC vs SSC

1. FSC measures cell size and surface area

2. SSC measure granularity/complexity/cytosolic structure

Uses of flow cytometry

1. Cell counting

2. Cell sorting

3. Analysis

   1. Immunophenotyping

   2. Functional assay