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Time and Money: Techniques for Neural Gene Expression Profiling

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TIME AND MONEY: TECHNIQUES FOR
NEURAL GENE EXPRESSION PROFILING
RAYNA M. HARRIS
HOFMANN LAB, THE UNIVERSITY OF TEXAS AT AU...

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RNAlater
qRT-PCRRNA-seq
O.C.T PFA
immunohistochemistryin situ
hybridization
Common molecular approaches for
neural gene ex...

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Time and Money: Techniques for Neural Gene Expression Profiling

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This was the very first talk I gave in front of a classroom based heavily on my research own personal research. It was 2013, and my audience was a group of students at the Marine Biological Lab in Woods Hole, MA and a group of students who Skyped in from Uruguay. The goal of this talk is to help students better understand when candidate gene approaches are preferred of whole genome approaches and vice versa.

You can watch this talk here: http://videocenter.mbl.edu/videos/video/630/in/channel/21/

This was the very first talk I gave in front of a classroom based heavily on my research own personal research. It was 2013, and my audience was a group of students at the Marine Biological Lab in Woods Hole, MA and a group of students who Skyped in from Uruguay. The goal of this talk is to help students better understand when candidate gene approaches are preferred of whole genome approaches and vice versa.

You can watch this talk here: http://videocenter.mbl.edu/videos/video/630/in/channel/21/

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Time and Money: Techniques for Neural Gene Expression Profiling

  1. 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/ 1
  2. 2. RNAlater qRT-PCRRNA-seq O.C.T PFA immunohistochemistryin situ hybridization Common molecular approaches for neural gene expression profiling 2
  3. 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) 3
  4. 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 4 qPCR Data Analysis
  5. 5. LMDTissue punches Increasing spatial resolution of RNA-seq and qRT-PCR RNAlater qRT-PCRRNA-seq 5
  6. 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) 6
  7. 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) 7
  8. 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. 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 9
  10. 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 10
  11. 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? 11
  12. 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 12
  13. 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 13
  14. 14. Acknowledgments 14

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