1. TIME AND MONEY: TECHNIQUES FOR
NEURAL GENE EXPRESSION PROFILING
RAYNA M. HARRIS
HOFMANN LAB, THE UNIVERSITY OF TEXAS AT AUSTIN
HTTP://VIDEOCENTER.MBL.EDU/VIDEOS/CHANNEL/21/
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2. RNAlater
qRT-PCRRNA-seq
O.C.T PFA
immunohistochemistryin situ
hybridization
Common molecular approaches for
neural gene expression profiling
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3. 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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4. 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
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qPCR
Data Analysis

5. LMDTissue punches
Increasing spatial resolution of
RNA-seq and qRT-PCR
RNAlater
qRT-PCRRNA-seq
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6. 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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7. 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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8. 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! 8

9. 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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10. 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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11. 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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12. 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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13. 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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14. Acknowledgments
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