HC 12 transcriptomics & proteomics

why look at the transcriptome?

changes in transcription may be connected to a phenotype. can help with diagnostics and mechanistic research

RNA expression varies by

• cell type

• development status

• cell-/disease status

• stimuli and drugs

• random noise

transcription techniques

• northern blotting

• quantitive PCR

• microarrays

• RNAseq

northern blotting

hybridization with DNA probes. detect transcriptase seze & isoform ratios. control for total amount of RNA loaded & sample intergrity

quantitive PCR

monitoring DNA aplification. SYBR add dye during PCR that becomes fluorescent when it binds to double-stranded DNA.

microarrays

hybridization with DNA probes. different techniques.

different techniques

• pins

• inkjet

• phototitho graphy

RNA-seq

samples of interest—> isolate RNA—> generate cDNA, fragment, size select, add linkers—> sequence end—> unsequenced RNA,RNA reads & short insert—> pre-mRNA short reads split by intron—> downstream analysis.

Based on next-gen seq technology

proteome

complete catalog of proteins in a organism.

• expressed throughout life

• expressed under all conditions

sup-proteome

• expressed in a cell or organelle

• expressed at a specific timepoint

• expressed under a specific condition

proteomics

large scale study of proteins

biochemical properties of a protein

size, hydrophobicity & charge