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TIME AND MONEY: TECHNIQUES FOR NEURAL GENE EXPRESSION

PROFILINGRAYNA M. HARRIS

HOFMANN LAB, THE UNIVERSITY OF TEXAS AT AUSTIN

HTTP://VIDEOCENTER.MBL.EDU/VIDEOS/CHANNEL/21/

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RNAlater

qRT-PCRRNA-seq

O.C.T PFA

immunohistochemistryin situ hybridization

Common molecular approaches for neural gene expression profiling

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Global gene expression profiling with RNA-seq

• RNA extraction (1-4 hrs)• Library prep (2 days)• Sequencing (3 days)• Bioinformatics (a few days)

• Filter low quality reads• Map to transcriptome• Identify differentially expressed genes

• Interpret data (months to years)

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Quantitative Real Time PCR

• Primer validation (weeks)• Store brains in RNALater or

homogenized in buffer• RNA Extraction (1-4 hrs)• cDNA synthesis (2 hrs)

– Oligo(dT) for mRNA only– Random hexamers for all RNA

• qPCR (3 hours)• Data analysis (~4 hours)

RNAlater

RNA isolation

cDNA synthesis

qPCRData Analysis

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LMDTissue punches

Increasing spatial resolution of RNA-seq and qRT-PCR

RNAlater

qRT-PCRRNA-seq

Tissue punches

• A. Freeze in O.C.T (5 min) and section (30 min/brain)

• B. Slice fresh tissue (5 min)• Punch regions of interest

(5min)• Homogenize and freeze tissue

(5 min)

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Laser Microdissection

• Freeze tissue in O.C.T (5 min)• Prepare membrane slides (20 min)• Histology (5 min/brain)• Laser microdissection

(~30min/brain)• RNA extraction (1-4 hrs)• Optional: RNA amplification

(1-2 days)

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Detecting RNA expression with

in situ hybridization• Riboprobe Synthesis (weeks)• Freeze brain in O.C.T. (5 min)• Section brains (hr/brain)• Hybridization & Detection (3 days)• Alternatives

– Radioactive– Fluorescent

• Visualize the signal – Count silver grains– Map distribution

Target RNA

Probe & RNA

Add HRP

Blue signal!

Detecting proteins with Immunohistochemistry

• Fix and cryoprotect tissue (2 days)• Section brains (~1hr/brain)• Bind primary and secondary

antibodies (3 days)• Visualize the signal • Quantify cell counts

or map distribution

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From the bench to publication

qPCR LMD ISH IHC$ spent $7,620 $4,161 $12,123 $6,695# papers 2 1 5 5$ per paper $3,810 $4,161 $2,425 $1,339

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A few questions that may help you choose most appropriate technique

• What are your molecules of interest? – mRNA or protein?– How soon after the stimulus will its activity be altered?

• How big is your experiment? – How many groups, animals, brain regions, genes/proteins?

• What resources do you have at your fingertips?– Core facilities and equipment– Validated PCR primers, riboprobes, antibodies?– A mentor who can help you collect & analyze the data?– Bioinformatic and statistical consulting?

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Candidate genes vs genomic approaches

• Histological approaches allow for co-localization• Histological approaches are low throughput• You may choose the wrong genes• Candidate genes act in networks that are poorly understand• Genomics allows systems level view of brain and behavior• Genomic approaches lack of spatial resolution

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Recommended Readings

IHC & ISH Mapping: Munchrath &Hofmann (2010) Distribution of Sex Steroid Hormone Receptors in the Brain of an African Cichlid Fish, Astatotilapia burtoni

Double IHC: O’Connell LA, Matthews BJ, Hofmann HA (2012) Isotocin regulates fatherhood in a monogamous cichlid fish.

qRT-PCR: Matz, Wright, Scott (2013) No Control Genes Required: Bayesian Analysis of qRT-PCR Data

Sequencing: https://wikis.utexas.edu/display/GSAF/Pricing

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Acknowledgments