Measuring Gene Expression

1. Measuring Gene Expression Chris Quirk, PhD Medical Sciences Program JH206, 856-2808 A501, CCQuirk

2. Innovations in Technology • Industrial revolution • 1750 – 1830 • Originated in Great Britain • Transformation from a largely rural population making a living almost entirely from agriculture to a town-centered society engaged increasingly in factory manufacture A501, CCQuirk

3. Innovations in Technology • Computer revolution (started during World War II) • Colossus I, a computer designed by the British to break Nazi military codes • Mark I, first fully automated ‘computer’ built by IBM for Harvard (calculator) • could store 72 numbers, each 23 decimal digits long. It could do three additions or subtractions in a second • a multiplication took 6 seconds, a division took 15.3 seconds, and a logarithm or a trigonometric function took over one minute A501, CCQuirk

4. Innovations in Technology • Genomics revolution • term “genome” was created by merging the words genes and chromosomes in 1920 • refers to all the DNA in an organism, including its genes • term “genomics” was coined in 1986 to describe the scientific discipline of mapping, sequencing, and analyzing genomes • name-sake to a scientific journal that was initiated at that time, Genomics • the goal of genomics is to make biological and functional sense of raw genetic information • In 2000, Francis Collins (NIH) and J. Craig Venter (Celera Genomics) announce a working draft of the human genome A501, CCQuirk

5. Human Genome Project • International effort began in October 1990 • Project was planned to last 15 years, but rapid technological advances accelerated the completion to 2003 • Project goals • Determine the complete sequence of the 3 billion DNA bases in the human genome • Identify all human genes • Make the genes accessible for further biological study • Genomes from other species also sequenced • Mice • Fruit flies • Zebra fish • Yeast • Nematodes • Plants • Many microbial organisms and parasites A501, CCQuirk

6. Transfer of genetic material: Structural changes in chromatin are activated Initiation of gene transcription Processing of emergent RNA transcript Transport of mature mRNA to cytoplasm Translation of the encoded polypeptide Central Dogma of Molecular Biology A501, CCQuirk

7. Basics of DNA • DNA is a long chain of molecules that takes on a double helix shape • adenine (A), guanine (G), thymine (T) and cytosine (C) • DNA is the genetic blue print for all cellular processes • Whereas DNA is the script for making RNA and proteins, RNA directs the production of all proteins A501, CCQuirk

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9. Breeding sheep: Short day vs. long day • Length of the estrous cycle 13 to 19 days, average 17 days • Phases of the estrous cycle are proestrus, estrus, metestrus, and diestrus • Estrus is the period of time when the ewe is receptive to the ram and will stand for mating (lasts approximately 24 to 36 hours) • Estrous cycles are usually affected by the seasons • The number of hours daily that light enters the eye of the animal affects the brain, which governs the release of certain precursors and hormones • Most sheep are seasonally polyestrus and short-day breeders • Begin to exhibit estrus when length of day begins decreasing • Most natural time for sheep to breed in the U.S. and Canada is the fall (Oct-Nov) A501, CCQuirk

10. What makes sheep short-day breeders? 14h light : 10h dark 10h light : 14h dark anestrus estrous cycling cells in hypothalamus A501, CCQuirk

11. Why does expression of p8 increase tumorigenic potential? C-LβT2 (express p8) p8-KD-LβT2 1.0 0.8 0.6 % tumor free C-LβT2 (n=13) p8-KD-LβT2 (n=22) censored 0.4 0.2 0.0 100 200 300 0 250 50 150 days post-injection A501, CCQuirk

12. Northern Blot Analysis p8-OE-LßT2 p8-KD-LßT2 • Developed in 1977 as standard method for detection and quantification of mRNA levels • provides a direct relative comparison of message abundance between samples on a single membrane • preferred method for determining transcript size and for detecting alternatively spliced transcripts • Limitations: • if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised • less sensitive than nuclease protection assays and RT-PCR • difficulty associated with multiple probe analysis C-LßT2 T3-1 LßT2 Kb 2.37 ßactin 18S 1.35 p8 CMV-p8 0.24 – – ++ + + p8 mRNA ~30,000 genes! A501, CCQuirk

13. Gene Expression Profiling • DNA Chips, Genome Chips, Bio chip, and cDNA arrays are all similar terms for one the most incredible and influential technologies in the area of genetic research known today – the GeneChip microarray • Affymetrix arrays are built using the same type of technology that is used to manufacture semiconductor chips, but rather than etching miniature circuits, Affymetrix builds millions of strands of DNA. A501, CCQuirk

14. When a single strand of DNA (ATCATG) matches a strand of RNA (UAGUAC), the two strands are "complementary" and will stick to each other. However, if the bases aren't complementary, they won't fit together. Affymetrix microarrays use base pairing attraction (hybridization) to help researchers identify what RNA sequences are present in a sample, and this then tells them how strongly those genes are being expressed. A501, CCQuirk

15. Build a short DNA strand — a probe — on the surface of a glass chip Sequence of the probe is compared to the rest of the human genome to make sure it doesn't match anywhere else For every probe, a mismatch probe is also produced Making a GeneChip… A501, CCQuirk

16. In all, there are 22 different probes, or data points, used to make sure that the microarray is detecting the correct piece of RNA By measuring that RNA with 11 probe pairs we can be absolutely certain that the gene we think is expressed actually is A501, CCQuirk

17. Features • The surface of the Affymetrix array is like a giant checkerboard that has been shrunk down to the size of a thumbnail. Each square (feature) on the checkerboard holds millions of copies of one unique type of probe • The most recent Affymetrix human genome array has more than 1.3 million squares/features • Represents about 47,000 different RNAs • Each feature on the array is about 11 microns across (~one-fifth the width of a human hair) • Affymetrix builds these probes one molecule at a time, using the same type of manufacturing technology that is used to build computer semiconductors • The molecules are built one layer (base) at a time, one stacked on top of another, like checkers A501, CCQuirk

18. Getting the RNA Ready • Extract RNA from the sample • Amplify it • Copying the RNA allows it to be more easily detected on the array • At the same time the RNA is copied, molecules of biotin (orange cups) are attached to each strand • Fragment the RNA A501, CCQuirk

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21. common genes genes unique to long day genes unique to short day Genome-wide arrays long day short day A501, CCQuirk

22. 600 500 400 Number of Unique Genes 300 200 100 0 1 GeneChip Number Genome-wide arrays long day short day A501, CCQuirk

23. 2 Genome-wide arrays long day short day 600 500 400 Number of Unique Genes 300 200 100 0 1 GeneChip Number A501, CCQuirk

24. 3 Genome-wide arrays long day short day 600 500 400 Number of Unique Genes 300 200 100 0 1 2 GeneChip Number A501, CCQuirk

25. 4 Genome-wide arrays long day short day 600 500 400 Number of Unique Genes 300 200 100 0 1 2 3 GeneChip Number A501, CCQuirk

26. Comparing Expression short day (cycling) long day (anestrus) • Researchers construct “heat maps”; graphical displays that color code gene expression • Increased expression is color coded in red • Decreased expression in blue 1 2 3 4 5 A501, CCQuirk

27. RNA Extraction Laboratory Organized by Christina Million Passe Kate Brannon Crystal White Laurel Bender JH209 & 211 A501, CCQuirk

28. RNA Isolation Protocol A501 - Techniques in Reproductive Diversity 28 November 2006 A501, CCQuirk

29. Step 1: Clean work area Notes: • RNA is very susceptible to degradation by ribonucleases (RNases) • RNases can be found almost everywhere in our environment, so we need to clean everything before we harvest RNA • RNase Away - noncarcinogenic RNase, DNase, and DNA decontaminant (proprietary formulation) Protocol: • Spray work area with 70% ethanol, then RNase Away, then ddH2O A501, CCQuirk

30. Step 2: Homogenize tissue in TRIzol Notes: • TRIzol - mixture of phenol and guanidine isothiocyanate (GTC) • Phenol - organic solvent that lyses cells and denatures proteins • GTC - inactivates RNases Protocol: • Add TRIzol to tissue sample and homogenize with a tissue homogenizer tip • Work quickly! Tissues will begin to release RNases as soon as you begin homogenizing! • Add additional TRIzol (total volume = 500 L) and incubate 5 minutes A501, CCQuirk

31. Step 3: Separate phases with chloroform Notes: • Chloroform - organic solvent that further denatures proteins and allows aqueous and organic phases to separate • Proteins stay in organic phase (bottom) • RNA goes to aqueous phase (top) • DNA goes to either, depending on pH • Low pH (like TRIzol), DNA stays in organic phase • High pH, DNA goes to aqueous phase • Phase Lock GelTM (PLG) - migrates between aqueous and organic phases upon centrifugation • Aqueous phase can be easily decanted A501, CCQuirk

32. Step 3: Separate phases (continued) Protocol: • Add chloroform to tissue homogenate, mix vigorousy, and incubate 3 minutes • During incubation, pellet gel in a PLG tube • Separate phases by centrifugation A501, CCQuirk

33. Step 4: Precipitate RNA Notes: • Nucleic acids are insoluble in concentrated alcohols Protocol: • Decant aqueous phase to a clean tube • Add 100% isopropanol, mix, and incubate 10 minutes • Pellet RNA by centrifugation A501, CCQuirk

34. Step 5: Wash RNA Notes: • Washing with less concentrated alcohols will remove impurities (residual salts, etc.) from RNA pellet • Alcohols must be diluted in RNase-free H2O • Diethyl pyrocarbonate (DEPC) is a histidine-specific alkylating agent that destroys enzymatic activity • Treating H2O with 0.1% DEPC inactivates RNases Protocol: • Remove supernatant • Wash pellet with ice cold 75% ethanol diluted in DEPC H2O • Pellet RNA by centrifugation A501, CCQuirk

35. Step 6: Redissolve RNA Notes: • Before redissolving, RNA must be dried to remove residual ethanol, which will make the pellet insoluble • Over-drying the pellet will make it difficult to dissolve • To avoid over-drying, we dry RNA until a “halo” (clear, dry circle) forms around the outside of the pellet, but the center remains white Protocol: • Remove as much supernatant as possible • Dry RNA until a “halo” forms • Redissolve in DEPC H2O and place on ice A501, CCQuirk

36. Steps 7-8: Determine RNA concentration & verify integrity Notes: • Intact RNA will yield two predominant bands when separated by electrophoresis, the 28S and 18S ribosomal subunits Protocol: • Determine RNA concentration according to the instructions of your group leader • Run 1 g RNA per lane on a 1% agarose + ethidium bromide gel and separate RNA by electrophoresis • Take a picture of your gel using the camera/UV box A501, CCQuirk

37. Things to do while you’re waiting for your gel to run… • Join Crystal as she performs mouse necropsies to collect organs of reproductive relevance • This option is completely voluntary! • Learn from Kate how we obtain our GeneChip data from the microarray data portal and see the types of things you can do with the data on the portal’s website • Sort through some of the Quirk Lab’s GeneChip data and see if you can find your favorite gene(s) • Read the “Red Book” protocol for RNA isolation • Current Protocols, Volume 1, Section 4.2.1 • Look over the TRIzol and PLG protocols • Find out how you can use these to obtain protein and DNA A501, CCQuirk