Genome Sciences

The Genome Sciences Facility offers a range of services for genomic, epigenomic and transcriptomic studies:

  • Consultations on experimental design & data validation
  • RNA, DNA, Protein quality control
  • Quantitative Real-Time PCR (SNP, CNV, mRNA, miRNA)
  • Gene expression microarrays (Illumina and Agilent)
  • SNP/CNV microarrays (Illumina)
  • Next Generation Sequencing, NGS (Illumina)
  • Data Analysis
Important instrumentation purchases and services have been made possible through Tobacco Settlement Funds (TSF), Juvenile Diabetes Research Foundation funds (JDRF), and through the Penn State Cancer Institute (PSCI) contract with the Department of the Navy.

 

Sample QA\QC

RNA Quality Assessment

Note: All RNA (total or mRNA/poly-A+) must be assessed for quality ("QCed") before use in array analysis. It is also recommended prior to any applications, as good quality RNA is the foundation of all subsequent work and you want to insure that you have quality and validity to your experiments.

  • At least 3 µl of RNA at a concentration > 0.1-0.5 µg/µl must be provided in a well-labeled tube for the quality control analysis.
  • The cost for RNA QC is $60 for up to 12 samples analyzed on RNA 6000 Nano chip and up to 11 samples analyzed for the RNA 6000 Pico chip (rare or micro-dissected samples).

Image of Perfect RNA - image of partially digested RNA -- image of severely degraded -- image of genomic DNA contaminated RNA

Acknowledgement and thanks for the Bioanalyzer images used

Protocols:

Links to additional protocols will be added as warranted.

Good information - An RNA Primer

We have found the Trizol/Tri-Reagent RNA Isolation Protocol to work well for many sample types.

Qiagen RNeasy kit is another preferred RNA isolation method and is available in the Core Supply Center (C1733).


RNA & DNA QC samples

RNA QC Samples to be submitted by filling out the proper form (RNA Nano, Pico ) or DNA located at

\\hersheymed\files\Research\CoreFacility\Results\Functional Genomics Incoming

and saving into the Functional Genomics Incoming QC Submission Folder on the network drive

\\hersheymed\files\Research\CoreFacility\Results\Functional Genomics Incoming\~QC Submissions.

NOTE: Nano chips hold 12 samples. Pico chips hold 11.

Map your network to this address:

\\hersheymed\files\Research\CoreFacility\Results\Functional Genomics Incoming   and into the QC Incoming folder

If you can not Print as a pdf, then use the Excel form  and File Save As _ into the folder above.

DNA samples are submitted following the same guidelines.

Samples are to be put in the freezer in C2705A in the Genome Sciences QC (Bioanalyzer) box. The freezer is marked with a Genome Sciences Dropoff sign.

HCAR researchers may place their samples in the box located in the Freeman lab, room 3200.

Note: Use your Name or other Unique description and please make sure the tubes are labeled with something descriptive, as well.

All incoming files to be saved into subfolders in the following folder:

\\hersheymed\files\Research\CoreFacility\Results\Functional Genomics Incoming

Completed results of all work are put in individual researcher folders located at

\\hersheymed\files\Research\CoreFacility\Results\Functional Genomics

Instrumentation

 Sample quantitation and quality control

qPCR
Microarray analyses
  • Axon 4000B microarray scanner
  • GE Healthcare Codelink Bioarray Parallel Processing System
  • Illumina BeadStation
  • Illumina BeadExpress
Next Generation Sequencing (NGS)
Software
  • Agilent Genespring
  • Illumina GenomeStudio
  • Illumina iCompute
  • Ingenuity Pathway Analysis
Additionally the facility contains refrigerators, -20ºC and -80ºC freezers, and associated small equipment (e.g. multichannel, electronic, and accordion pipettes).

New  Nucleic acid isolation is now available using the QIAsymphony DNA/RNA Extraction Robot.  The QIAsymphony SP enables sample preparation of DNA, RNA, bacterial and viral nucleic acids from whole blood, saliva and buffy coat among other sample types. 

FEES per sample:

200 ul Whole Blood DNA prep  $      5.90
400 ul Whole Blood  DNA prep  $      7.40
1000 ul Whole Blood DNA prep  $     10.80
200 ul DNA prep buffy coat  $      5.90
400 ul DNA prep buffy coat  $      7.40
400 ul RNA prep  $      6.40
800 ul RNA prep  $     12.00
PAXgene_RNA  $     12.00
1000 ul Saliva DNA prep   $     10.80

For more information, contact Sue Patrick x5676  sdp8@psu.edu

 

NEW  Covaris Adaptive Focused Acoustics Ultrasonicator providing DNA and chromatin shearing capabilities, is now available in room C2706.  The Covaris enables shearing of samples without thermal damage.  The equipment is available on a sign-up basis. Fees: $8.00/per sample (includes specialty tube).  Please contact Sue Patrick x5676 sdp8@psu.edu for more details.

 

NextGen Sequencing

Genome Sciences has added new Illumina sequencing instrumentation (MiSeq and HiSeq 2500) for both focused and large-scale sequencing by synthesis. MiSeq and HiSeq 2500  services are now available. Specific library construction services will be available in early 2013. Contact Genome Sciences staff at x5823 for more information.

MiSeq:
For library construction we recommend Illumina library preparation kits, and most other library preparation kits with Illumina adaptors will work with the MiSeq. To aid in choosing the appropriate library construction kit see our compatibility chart. If you have a sequencing project please consult Genome Sciences staff before submitting samples.
Single Read (50bp) - $925
Paired-end Read (150x150bp) – $1,180
Paired-end Read (250x250bp) - $1,270
 
HiSeq 2500:
Both rapid run and high output workflows are now available on the HiSeq2500.
50 Single Read High Output  - $936.00
100 Single Read High Output - $1,425.00
200 Paired Read High Output - $2,536.00
50 Single Read Rapid Output - $2,856.00
200 Single Read Rapid Output - $3,293.00 

qPCR

Please remember to check News & Events and the Calendar for up-to-date information.

Quantitative Real-Time PCR

 For researchers at the Hershey Center for Applied Research we are trying to make things easy for you.

  • The SDS2.2.2 software is on a computer in the Freeman lab, room 3200.
  • 384-well plates and covers may be be available in room 3200.  They may also be purchased through the Supply Center.

Sign onto the computer with your e-pass -or the person currently signed on lets you create your file.

You will set up the plate as if you are running it on the instrument, and type the barcode into the initial window that you would normally scan into.

NOTE: If you add a new detector, you will need to export that so it can be uploaded onto instrument computer. Call  x5823 or email Rob  or Georgina for instructions.

Save the SDS file you create into the "Incoming 7900 Plates" folder in the network folder located here:

\\hersheymed\files\Research\CoreFacility\Results\Functional Genomics Incoming

 

For 384 well plates : Save as -- YourName_PI_BARCODE.

 

The plate is then put in the refrigerator in room 3200 and we collect them once a day around 1:30 PM.

NOTE: if you miss the 1:30 deadline, the plate can wait until the next day and/or you can bring it over to College of Medicine, room C2705 by 4:30.


---- Create the file and save it on the Functional Genomics Incoming folder on the network drive. We pull that file up and run the plate and put the file - now with data - back onto the network in the Functional Genomics Results folder in the individual researchers' folder the next day for you to analyze (again, on the computer in Freeman lab / 3200).


Quantitative Real-Time PCR is used to very precisely measure the amount of gene expression in cells or tissue of interest, relative to an endogenous control gene such as 18s, b-actin, etc. The two major methods to perform QRTPCR that are supported here in the Functional Genomics Core Facility are dual-labeled probe chemistry (TAQMAN®) and SYBR green chemistry.

Our recommendation for ease of use is to utilize the pre-optimized Primer/Probe Applied Biosystems Gene Expression Assays ( aka Assays-on-Demand ). These may be ordered through the Core Facility.

We can assist in finding other sources should your gene not be available at ABI, either with another company or with design assistance ( see below on this page ).

A very good source of information describing techniques and including troubleshooting may be found in the 2006 GenomeTech-RT-PCR TechGuide

A brief description ( of both Taqman 5'-nuclease ( dual-labelled probe chemistry ) and SYBR green ) follows:

Taqman Dual-Labeled Probe ( 5'-nuclease )

An oligo that is complementary to a portion of your gene of interest sequence between your PCR primers. This oligo has an fluor attached to the 5' end, and a quencher attached to the 3' end. When the oligo has hybridized to your gene sequence, and the polymerase incorporates it into the new product, the fluor is allowed to distance itself from the quencher. The rate at which new copies of your gene of interest are generated is inferred by the rate at which the intensity of this free fluor increases.

Dual labeled probes are more specific than SYBR green because dual labeled probe chemistry requires specific amplification of the gene of interest in order for fluorescence to be generated. SYBR Green, however, will generate fluorescent signal in the cases of mispriming and the formation of primer dimers. In addition, two different genes of interest may be amplified in one reaction, and detected independently from one another by using different fluors one each dual labeled probe. The disadvantage of using dual-labeled probes is that one dual-labeled probe must be purchase for each gene of interest.

SYBR Green

A dye that will bind to double stranded nucleic acid. As your primers anneal and the polymerase extends to make new copies of your gene of interest, the amount of SYBR green increases proportionately. The rate at which new copies of your gene of interest are generated is inferred by the rate at which SYBR green fluorescence increases.

Should you be using an unavailable species and/or your gene is not yet listed we are also available to assist with Primer Design and Purchase.

A frequently updated list of validated primer/probe sets for quantitation of a variety of mRNAs is maintained at http://www.realtimeprimers.org/

To design your own primer/probe sets, you can use Primer Express, which is available in our facility (C2705).

Contact Rob Brucklacher  (x5823) for more information and assistance.

Flanking 5' (forward) and 3' (reverse) Primers can be made in our Macromolecular Core Facility -- or through external companies such as IDTDNA, Invitrogen, Applied Biosystems, etc.

Microarrays

 Gene Expression Microarrays

The Genome Sciences Core uses the Illumina platform which comes in several formats

·         Mouse Whole Genome Array (multiple of 6) - $345/array

·         Mouse RefSeq8 Array (multiples of 8, only sequences are RefSeq ) - $250/array.

·         Human HT_12 arrays (multiples of 12 ) - $220/array.

Genotyping Microarrays

·         For focused genotyping/CNV studies the VeraCode platform using the BeadXpress instrument is extremely powerful. This platforms lets investigators choose their specific SNPs of interest in 48-, 96-, 144-, 192-, or 384-plex formats. Contact the Core for more information.

 

Recent Publications

1. Abcouwer SF, Lin C-M, Wolpert EB, Shanmugam S, Schaefer EW, Freeman WM, Barber AJ, Antonetti DA (2010).Vascular permeability and apoptosis are separable processes in retinal ischemia-reperfusion injury: Effects of ischemic preconditioning, bevacizumab and etanercept. IOVS. Jun 16. Epub ahead of print.
2. Balliet, RM, Chen, G, Gallagher, CJ, Dellinger, RW, Sun, D, and Lazarus, P (2009). Characterization of UGTs active against SAHA and association between SAHA glucuronidation activity phenotype with UGT genotype. Cancer Res 69, 2981-2989.
3. Barthéléry M, Salli U, Freeman WM, Vrana KE (2009). 2-DIGE identification of differentially expressed hNRNPs and transcription factors during neural differentiation of human ES cells Proteomics: Clinical Applications. 3:505-514.
4. Bayerl, MG, Bruggeman, RD, Conroy, EJ, Hengst, JA, King, TS, Jimenez, M, Claxton, DF, and Yun, JK (2008). Sphingosine kinase 1 protein and mRNA are overexpressed in non-Hodgkin lymphomas and are attractive targets for novel pharmacological interventions. Leuk Lymphoma 49, 948-954
5. Blake, DC, Jr., Mikse, OR, Freeman, WM, and Herzog, CR (2010). FOXO3a elicits a pro-apoptotic transcription program and cellular response to human lung carcinogen nicotine-derived nitrosaminoketone (NNK). Lung Cancer 67, 37-47.
6. Blevins-Primeau, AS, Sun, D, Chen, G, Sharma, AK, Gallagher, CJ, Amin, S, and Lazarus, P (2009). Functional significance of UDP-glucuronosyltransferase variants in the metabolism of active tamoxifenmetabolites. Cancer Res 69, 1892-1900.
7. Bortner, JD, Jr, Das, A, Umstead, TM, Freeman, WM, Somiari, R, Aliaga, C, Phelps, DS, and El-Bayoumy, K (2009). Down-regulation of 14-3-3 isoforms and annexin A5 proteins in lungadenocarcinoma induced by the tobacco-specific nitrosamine NNK in the A/J mouse revealed by proteomic analysis. J Proteome Res 8, 4050-4061.
8. Bowyer JF, Pogge AR, Delongchamp RR, O’Callaghan JP, Patel KM, Vrana KE, Freeman WM (2007). A threshold neurotoxic amphetamine exposure inhibits synaptic plasticity gene expression in the parietal cortex. Neuroscience. 144:66-76.
9. Brucklacher RM, Patel KM, Bixler GV, VanGuilder HD, Antonetti DA, Lin C-M, LaNoue KM, Barber AJ, Gardner TW, Bronson SK, Freeman WM (2008). Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response. BMC Medical Genomics. 1:26.
10. Chen, G, Blevins-Primeau, AS, Dellinger, RW, Muscat, JE, and Lazarus, P (2007). Glucuronidation of nicotine and cotinine by UGT2B10: loss of function by the UGT2B10 Codon 67 (Asp>Tyr) polymorphism. Cancer Res 67, 9024-9029.
11. Chen, G, Dellinger, RW, Gallagher, CJ, Sun, D, and Lazarus, P (2008). Identification of a prevalent functional missense polymorphism in the UGT2B10 gene and its association with UGT2B10 inactivation against tobacco-specific nitrosamines. Pharmacogenet Genomics 18, 181-191.
12. Chen, G, Dellinger, RW, Sun, D, Spratt, TE, and Lazarus, P (2008). Glucuronidation of tobacco-specific nitrosamines by UGT2B10. Drug Metab Dispos 36, 824-830.
13. Ding, W, Mouzaki, M, You, H, Laird, JC, Mato, J, Lu, SC, and Rountree, CB (2009). CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis. Hepatology 49, 1277-1286.
14. Elftman, MD, Norbury, CC, Bonneau, RH, and Truckenmiller, ME (2007). Corticosterone impairs dendritic cell maturation and function. Immunology 122, 279-290.
15. Feith, DJ, Bol, DK, Carboni, JM, Lynch, MJ, Sass-Kuhn, S, Shoop, PL, and Shantz, LM (2005). Induction of ornithine decarboxylase activity is a necessary step for mitogen-activated protein kinase kinase-induced skin tumorigenesis. Cancer Res 65, 572-578.
16. Feith, DJ, Shantz, LM, Shoop, PL, Keefer, KA, Prakashagowda, C, and Pegg, AE (2007). Mouse skin chemical carcinogenesis is inhibited by antizyme in promotion- sensitive and promotion-resistant genetic backgrounds. Mol Carcinog 46, 453-465.
17. Fort PE, Freeman WM, Kimball SR, Singh RSJ, Gardner TW. The retinal proteome in experimental diabetic retinopathy: Upregulation of Beta and Gamma-crystallins and reversal by insulin (2009). Mol. Cell. Proteomics. 8:767-779.
18. Freeman WM, Lull ME, Patel KM, Brucklacher RM, Morgan D, Roberts DCS, Vrana KE (2010). Gene expression changes in the mesolimbic pathway following abstinence from cocaine self-administration. BMC Neuroscience 2010; 11:29.
19. Freeman WM, Bixler GV, Brucklacher RM, Lin C-M, Patel KM, VanGuilder HD, LaNoue KM, Barber AJ, Antonetti DA, Gardner TW, Bronson SK (2009). A novel multi-step evaluation process of preclinical drug development biomarkers. The Pharmacogenomics Journal. Dec 8. Epub ahead of print.
20. Freeman WM, Bixler GV, Brucklacher RM, Kimball SR, Jefferson LS, Bronson SK. Transcriptomic comparison of the retina in two mouse models of diabetes (2009). Journal of Ocular Biology, Diseases, and Informatics. 2:202-213.
21. Freeman WM, Patel KM, Lull ME, Erwin MS, Bruchlacker RM, Morgan D, Roberts DCS, Vrana KE (2008). Persistent alterations in mesolimbic gene expression with abstinence from cocaine self-administration. Neuropsychopharmacology. 33:1807-1817.
22. Gallagher, CJ, Ahn, K, Knipe, AL, Dyer, AM, Richie, JP, Jr, Lazarus, P, and Muscat, JE (2009). Association between haplotypes of manganese superoxide dismutase (SOD2), smoking, and lung cancer risk. Free Radic Biol Med 46, 20-24.
23. Gallagher, CJ, Kadlubar, FF, Muscat, JE, Ambrosone, CB, Lang, NP, and Lazarus, P (2007). The UGT2B17 gene deletion polymorphism and risk of prostate cancer. A case-control study in Caucasians. Cancer Detect Prev 31, 310-315.
24. Gallagher, CJ, Muscat, JE, Hicks, AN, Zheng, Y, Dyer, AM, Chase, GA, Richie, J, and Lazarus, P (2007). The UDP-glucuronosyltransferase 2B17 gene deletion polymorphism: sex-specific association with urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronidation phenotype and risk for lung cancer. Cancer Epidemiol Biomarkers Prev 16, 823-828.
25. Hodge DL, Yang J, Buschman MD, Schaughency PM, Dang H, Bere W, Yang Y, Savan R, Subleski JJ, Yin XM, Loughran TP Jr, Young HA. Interleukin-15 enhances proteasomal degradation of bid in normal lymphocytes: implications for large granular lymphocyte leukemias. Cancer Res. 2009 May 1;69(9):3986-94.
26. Iwona Hirschler-Laszkiewicz, Qin Tong, Kathleen Conrad, Wenyi Zhang, Wesley W. Flint, Alistair J. Barber, Dwayne L. Barber, Joseph Y. Cheung, and Barbara A. Miller TRPC3 Activation by Erythropoietin Is Modulated by TRPC6. J. Biol. Chem. 2009 284: 4567-4581.
27. Jaishankar A, Barthéléry M, Freeman WM, Salli U, Vrana KE (2009). Human Embryonic and Mesenchymal Stem Cells Express Different Nuclear Proteomes. Stem Cells Develop. 18:793-802.
28. Kline, CL, Jackson, R, Engelman, R, Pledger, WJ, Yeatman, TJ, and Irby, RB (2008). Src kinase induces tumor formation in the c-SRC C57BL/6 mouse. Int J Cancer 122, 2665-2673.
29. Kuntz-Melcavage, KL, Brucklacher RM, Grigson PS, Freeman WM, Vrana KE (2009). Gene expression changes in a heroin behavioral incubation model. BMC Neuroscience. 10:95.
30. Olson, KC, Dellinger, RW, Zhong, Q, Sun, D, Amin, S, Spratt, TE, and Lazarus, P (2009). Functional characterization of low-prevalence missense polymorphisms in the UDP-glucuronosyltransferase 1A9 gene. Drug Metab Dispos 37, 1999-2007.
31. Origanti, S, and Shantz, LM (2007). Ras transformation of RIE-1 cells activates cap-independent translation of ornithine decarboxylase: regulation by the Raf/MEK/ERK and phosphatidylinositol 3-kinase pathways. Cancer Res 67, 4834-4842.
32. Pastor, DM, Irby, RB, Poritz, LS (2010). Tumor necrosis factor alpha Induces p53 up-regulated modulator of apoptosis expression in colorectal cancer cell lines. Dis Colon Rectum 53, 257-63.
33. Prokopczyk, B, Sinha, I, Trushin, N, Freeman, WM, and El-Bayoumy, K (2009). Gene expression profiles in HPV-immortalized human cervical cells treated with the nicotine-derived carcinogen 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone. Chem Biol Interact 177, 173-180.
34. Roff, A, and Wilson, RT (2008). A novel SNP in a vitamin D response element of the CYP24A1 promoter reduces protein binding, transactivation, and gene expression. J Steroid Biochem Mol Biol 112, 47- 54.
35. Salli, U, Fox, TE, Carkaci-Salli, N, Sharma, A, Robertson, GP, Kester, M, and Vrana, K (2008). Propagation of undifferentiated human embryonic stem cells with nano-liposomal ceramide. Stem Cells Dev 18, 55-65.
36. Shah, MV, Zhang, R, Irby, R, Kothapalli, R, Liu, X, Arrington, T, Frank, B, Lee, NH, and Loughran, TP, Jr (2008). Molecular profiling of LGL leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes. Blood 112, 770-781.
37. Spidel, JL, Wilson, CB, Craven, RC, and Wills, JW (2007). Genetic studies of the beta-hairpin loop of Rous sarcoma virus capsid protein. J Virol 81, 1288-1296.
38. Sudol, M, Tran, M, Nowak, MG, Flanagan, JM, Robertson, GP, and Katzman, M (2009). A nonradioactive plate-based assay for stimulators of nonspecific DNA nicking by HIV-1 integrase and other nucleases. Anal Biochem 396, 223-230.
39. VanGuilder HD, Brucklacher RM, Patel K, Ellis RW, Freeman WM, Barber AJ. (2008) Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retina. Eur J Neurosci. 28:1-11.
40. Van Guilder HD, Vrana KE, Freeman WM (2008). 25 years of quantitative PCR for gene expression analysis. BioTechniques. 44: 619-626.
41. Vunta, H, Belda, BJ, Arner, RJ, Channa Reddy, C, Vanden Heuvel, JP, and Sandeep Prabhu, K (2008). Selenium attenuates pro-inflammatory gene expression in acrophages. Mol Nutr Food Res 52, 1316-1323.
42. Yang, J, Liu, X, Nyland, SB, Zhang, R, Ryland, LK, Broeg, K, Baab, KT, Jarbadan, NR, Irby, R, and Loughran, TP, Jr (2009). Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway. Blood 115, 51-60.
43. You, H, Ding, W, Rountree, CB (2010). Epigenetic regulation of cancer stem cell marker CD133 by transforming growth factor-beta. Hepatology. 52:945-5329.
44. Zhang B, Ramesh G, Uematsu S, Akira S, Reeves WB. TLR4 signaling mediates inflammation and tissue injury in nephrotoxicity (2008). J Am Soc Nephrol. 19:923-32.
45. Zheng, Y, Sun, D, Sharma, AK, Chen, G, Amin, S, and Lazarus, P (2007). Elimination of antiestrogenic effects of active tamoxifen metabolites by glucuronidation. Drug Metab Dispos 35, 1942-1948.

Calendar

For experimental scheduling and Core closures please see:

http://genomics.calendarhost.com/cgi-bin/calweb/calweb.cgi

Contact

The Genome Sciences Core is located in C2705.

Director: 

Willard M. Freeman, Ph.D.

Email: wfreeman@psu.edu

Staff:

Robert Brucklacher, B.A., B.S., Rbrucklacher@hmc.psu.edu

Gerogina Bixler, B.A., GBixler2@hmc.psu.edu

Phone: 717-531-5823