Techniques in Molecular Biology

N. L. Naeger BeeSpace annual workshop Wed., July 9, 2008

Review from yesterday

Goals of BeeSpace
Analyze N vs. N in bee social behavior Measure gene expression Develop computational tools

mRNA captures both nature and nurture In bees, theres a robust relationship between gene expression and behavior

Today
Methods to analyze gene expression
qRT-PCR
Quantitative real time polymerase chain reaction Gives an exact quantity of mRNA of a single gene

In situ analysis
Shows localization of gene expression

Microarray
Looks at gene expression for many genes at the same time

qRT-PCR
The first steps: Isolate the tissue of interest
Keep it cold! Freeze drying

Extract the RNA

RNA extraction
Different solubilities
Homogenize tissue Chloroform/phenol vs. water Nucleic acids stay in water

Guanidium thiocyanate

Precipitate with ethanol Trap nucleic acids on a filter Digest with DNase Wash and rinse, resuspend in water

Spectrophotometry
Different molecules absorb or reflect different wavelengths of light Spectrophotometers measure the amount of specific wavelengths of light Nucleic acids show maximal absorbance at 260nm, proteins 215-230nm, etc.

cDNA synthesis
DNA is more stable than RNA PCR must be done on DNA We must make a DNA copy (cDNA) from the RNA original

Retrovirus

PCR
Heat to separate strands Primers to give the polymerase a starting point
Primers give gene specificity

Replicate strand from individual nucleotides, A C T and G

qRT-PCR
Combination PCR thermocycler and spectrophotometer Measure absorbency after each round of replication Use known quantities as markers

In situ analysis
Create a complimentary strand to your RNA Label that probe with a visible marker Use chemical techniques to get the probe into the tissues of interest, and then wash away the excess The higher the gene expression, the better visibility of the marker

Microarray
Look at the expression of many genes (sometimes the entire genome) all at once Use a robotic arm to affix tiny dots of complimentary DNA onto a slide Wash RNA over the slide and allow it to hybridize Analyze the color pattern by computer

Yes, biology, but.

A lot of chemistry Physics Math and statistics English and writing
If its not published, its as if the work was never done

Reading and the library Computers