All of the RNA in a cell

(mRNA, tRNA, and other non-coding RNA)

• Cell type
• Developmental Stage
• External Stimuli

several techniques for measuring RNA abundance rely on hybridization

• btwn complementary DNA strands 
• OR btwn comp strands of DNA and RNA

Microarray & Northern Blot have similar principles.

Whats the main difference?

• Electrophoresis of RNA through gel
• Blot to transfer RNA to solid support
• Nylon or nitrocellulose membrane
• Incubate with radioactive probe
• Intensity of hybridization signal proportional to amount of RNA

• Be sure RNA quality is good, not degraded
• ---degraded RNA? Hybridization won't reflect quantity present
• Use more probe than RNA complementary to it
• ---too little probe, amt of RNA bound may not reflect amount in sample (could be more in it)
• Allow enough time for hybridization to occur btwn probe and target RNA
• ---not enough time? some target RNA wont bind.
• Be sure each sample has the comparable amounts of RNA. Use loading control.

Northern Blot

Target

Probe

Loading Control

• Target: RNA of interest on the gel
• Probe: Labelled oligonucleotide sequence that bind the target DNA
• Loading Control: transcript expressed equally in all cells that's used to check amount of RNA loaded on gel (ubiquitin)

NB: Limited by # of probes that can be used on the same blot (no more than 3) and # of lanes

Micrarray: RNA levels of every gene in the genome analyzed in parallel (1000s of genes)

More on Microarrays

DNA? RNA?

DNA attached to plate

RNA labelled

• usually indirectly (synthesize labelled cDNA intermediate)
• bound DNA is the "probe"
• Labelled RNA or cDNA is "target"
• bound probe present in excess

• Label each sample with a different fluorophore
• these compete to hybridize to DNA probe
• Record ratio btwn two

Examples...

• Tumor vs normal tissue
• field site 1 vs 2
• embryo vs adult

Spotted Arrays and Photolithography

Spotted arrays:

• DNA mechanically placed on glass slide
• Nedd to deliver DNA in nanoliter to picoliter volumes bc too small for normal pipets
• Robot "prints/spots" DNA in precise locations on glass slide
• Robot spotter ised to place DNA has multiple pins that act by capillary action (like fountain pen)
• Pins drop into micrometer plate containing qPCR amplified cDNA or genome DNA
• Oligonucs always spotted this way

• Pins dipped into DNA solution in microtiter wells
• Robot remove pins with DNA to slides
• Robot prints DNA onto slide (stuck by hydrostatic interactions)
• same spots usually printed at different locations
• pins washed btwn printing rounds
• Hundreds of slides could be printed in a day

Ink jet printer microarrays

(old approach along with mechanical spotting)

• inkjet printhead drew up DNA
• Printhead moved to specifi location on solid support
• DNA ejected through small hole
• Devoed by Agilent tec

+ cheap

+allowed one to study genes that hadnt been sequenced (used ESTs to discover new genesr to study)

-wasnt as sensitive as other techniques

-amount & quality of DNA deposited varied. Made it difficult to get results.

-xould only get info on relative gene expression

• IJ printheads synthesize DNA oligonucelotides on a glass slide
• printhead moves to location and specific nucleotide type is ejected
• a chem rxn allows nucs to attach. Repeat 60x
• Result> oligo strand with 60 bases
• Agilent tech again

--same process used to print comp circuits

--oligo nuceotides synthesized on silicon chip one base at a time
--Affymetrix gene chips & nimblegen

-Oligonucleotides synthesized

        Usually 20-25 bp in length

        10-20 diff oligonucleotides for each gene

-Olilgonucleotides for each gene selected by computer program to be the following:

        Unique in genome

        Non-overlapping (to prevent cross-hybridization)

        Devoed to have specific G-C content and reduce likelihood of forming hairpin loops

Light-activated chemical reaction

--allows addition of bases to growing oligonucleotide

Custom masks

--prevent light from reaching spots where the bases not wanted

• Want to add nucleotide G
• Mask all other spots on chip
• Light shines only where addition of G is desired
• G added and reacts
• Now G is on subsets of oligonucleotides

Example: Adding a second base

• Want to add a T New mask covers spots where T not wanted
• Light shines on mask
• T added
• Continue for all four bases
• Need 80 masks to make 20-mer oligonulceotide

• Also relies on photolithography
• Uses mirrors (300,000-720,00 instead of masks)
• Cheaper...dont have to make masks, so can afford to make longer oligonucleotides

Two-channel hybridization

(competitive)

-using reverse transcriptase to make single stranded cDNA

-Fluorescently labelled nucleotides incorporated as cDNA synthesized

-Popular labels: Cyanine 3 (Cy3) and Cyanine 5 (Cy5). Each fluoresces at a different wavelength

-Maintains a constant temperature and mixes labeled target with probes

-nimblegen mixers attach to side, volume of fluid ot hybridize is low

**allows you to conserve sample and ensure uniform siganl and high sensitivty in microarray

-double stranded cDNA made with Rev Trans

-linker added with T7 RNApolym recognition site

-T7 RNA polymerase added and biotin labeled RNA bases (UTP-biotin)

-Biotin label incorporated into cRNA

-production of cRNA amplifies the original RNA population

-used by Affymetrix

A DNA Microarray Experiment