Testing novel fluorescent in situ hybridization methods

Udo Onwubiko and Nick DeeMethods Development

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The purpose of this project - To improve the quality of fISH experiments by investigating the quality of a fluorescing compound that can be used for FISH experiments.

Compound: A protein model from the baker lab at UWName: dig-GFP- reported to have a very strong affinity to digoxigenin( hapten used often in fISH experiments)

Introduction

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To evolve experimental procedures that may help to producing more better quality data, such as providing a quantitative readout of gene expression by fluorescent in situ hybridization (dFISH)

An ongoing process!

Project Goals

DESIGN

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Table 3Peak 1 (0.4 mg/mL)

Conc 1 0.2 µg/mL = 5 µl in 10mls TNB

Conc 2 0.5 µg/mL = 12.5 µl in 10mls TNB

Conc 3 1.0 µg/mL = 25 µl in 10mls TNB

Conc 4 2.0 µg/mL = 50 µl in 10mls TNBConc 5 4.0ug/ml 100ul in 10mls TNB

Peak 2 (1.8 mg/mL)

Conc 1 0.2 µg/mL = 2.2 µl in 20mls TNB

Conc 2 0.5 µg/mL = 5.6 µl in 20mls TNB

Conc 3 1.0 µg/mL = 11 µl in 20mls TNB

Conc 4 2.0 µg/mL = 22 µl in 20mls TNBConc 5 4.0ug/ml = 44ul in 20mls TNB

The experiment is designed as follows--Two different protein stocks with different concentrations were used during this experiment. -Protein stocks: PEAK1 PEAK2-Concentrations: 0.4mg/ml 1.8mg/ml-To observe fluorescence, a FISH experiment is carried out.-The RNA probes are : Drd1a, Etv1, Calb1 and a blank(no probe)-A portion of each protein stock was diluted and divided into different concentrations for the FISH procedureFormula: C1*V1=C2*V2The table above shows the varying stock concentrations for each of the protein stocks used.

fISH procedure

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EXPECTATIONS

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Calb1 control slide- Expected observationPicture credit: Chris Hill

DAPICalb1

RESULTS

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Dig-GFPDAPI

mRNA target: Calb1Peak 2, conc. 4ug/ml

2nd dig-gfp run: varied conc. & times

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3 slidesPeak 2 DIG-GFP Conc. #1 20.0 µg/mL = 33 µl in 3mls PBS

3 slidesPeak 2 DIG-GFP Conc. #2 50.0 µg/mL = 82.5 µl in 3mls PBS

3 slidesPeak 2 DIG-GFP Conc. #3 100.0 µg/mL = 165 µl in 3mls PBS

3 slidesPeak 2 DIG-GFP Conc. #4 400.0 µg/mL = 660 µl in 3mls PBS

2 slides Pooled DIG-GFP 1:10 Dilution = 200 µl in 1800 µl PBS

2 slides Pooled DIG-GFP 1:4 Dilution = 500 µl in 1.5 mls PBS

2 slides Pooled DIG-GFP 1:2 Dilution = 1ml in 1ml PBS

A

200-128614b.3.2 200-128614b.2.3 200-128614b.3.3     1 1 1

Conc 1=20ug/ml Conc 1=20ug/ml Conc 1=20ug/mlA01 45minA02 1.5hrA03 2.0hr

B

200-128614b.4.2 200-128614b.4.3 200-128614b.5.3     1 1 1

Conc 2=50ug/ml Conc 2=50ug/ml Conc 2=50ug/mlB01 45minB02 1.5hrB03 2.0hr

C

200-128614b.5.2 309-F0508b.1.3 309-F0508b.2.3      1 1 1

Conc 3=100ug/ml Conc 3=100ug/ml Conc 3=100ug/mlC01 45minC02 1.5hrC03 2.0hr

D

200-128614b.1.3 309-F0508b.3.3 309-F0508b.4.3            

1 1 1Conc 4=400ug/ml Conc 4=400ug/ml Conc 4=400ug/ml

D01 45minD02 1.5hrD03 2.0hr

E

309-F0508b.5.3 309-F0508b.1.4 309-F0508b.2.4      1 1 1

Pooled- 1:10 Pooled- 1:4 Pooled- 1:2E01 1hrE02 1hrE03 1hr

F

309-F0508b.3.4 309-F0508b.4.4 309-F0508b.5.4            

1 1 1Pooled- 1:10 Pooled- 1:4 Pooled- 1:2

F01 1hrF02 1hrF03 1hr

Results (2)

9Calb1 expressionPeak 2, C2. 400ug/ml

Calb1DAPI

Calb1DAPI

DAPI

CONCLUSION

• At a concentration of 400ug/ml, we see specs of green fluorescence carried by the dig protein with affinity for digoxigenin

• No signal was seen at concentrations between the ranges of 100ug/ml 4ug/ml and 50ug/ml

• The presence of GFP is independent on the amount of time allowed after the addition of the protein

• The data suggests that there is a correlation between the amount and concentrations of dig-GFP added and the amount of green fluorescence observed

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• Amy Bernard - Awesome Mentor• Special thanks to Nick Dee- You definitely helped make

this internship a success!• All Methods Development groups • The Allen Institute

Acknowledgements

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• Chris Hill- Image : Calb1 gene expression, Allen Institute for Brain Science 2014

• 2. Collection of resources available via the Allen Brain Atlas data portal Website: ©2014 Allen Institute for Brain Science. Allen Brain Atlas [Internet]. Available from: http://www.brain-map.org