gene expression analysis

Northern blotting

Technique for detecting specific RNA sequences.

RT-PCR

Reverse transcription followed by polymerase chain reaction.

Microarray

Analyzes expression of thousands of mRNAs simultaneously.

Western blotting

Technique for detecting specific proteins in samples.

Radioactive probes

Used for nucleic acid detection via autoradiography.

32P

High energy radioactive isotope, sensitive detection.

35S, 14C, 3H

Lower energy isotopes, less sensitive, higher resolution.

Chemical probes

Antigen linked to DNA, detected by enzyme-linked antibodies.

Fluorescent probes

Various compounds used for detection with specialized equipment.

Reverse transcriptase

Enzyme that synthesizes DNA from an RNA template.

cDNA synthesis

Conversion of mRNA into complementary DNA.

PCR amplification

Process of exponentially increasing DNA copies.

Gel electrophoresis

Technique for separating nucleic acids or proteins by size.

RNA-seq

Method for analyzing expression of many genes.

Cluster analysis

Identifies sets of coordinately regulated genes.

In situ hybridization

Determines expression locations of specific genes.

Epigenetics

Heritable changes in gene activity without DNA sequence changes.

DNA methylation

Chemical modification affecting gene expression regulation.

3rd generation sequencing

Single molecule sequencing with real-time detection.

PACBio

Uses fluorescently labeled dNTPs for DNA sequencing.

Oxford nanopore

DNA sequencing through protein nanopores with electric current.

3 types of probes used for measuring nucleic acid levels

radioactive, chemical, fluorescent

RT-PCR process

RT added to tube containing isolated mRNAs - RT makes first DNA strand - mRNA degraded by RNAse - DNA polymerase makes second DNA strand using primer - results in cDNA

use of DNA microarray

assays compare patterns of gene expression in different times/tissues/conditions

PACBio process

dna fragmented and ligated to adapters & DNA polymerase - fluorescent dNTPs added - light emitted from excited fluorophore detected -- records wavelength and relative base position

oxford nanopore steps

thousands of protein nanopores in synthetic membrane - tethering proteins bring dna towards these nanopores - motor protein unwinds dsDNA - electric current drives DNA through pore and causes characteristic disruptions to the current