The full-service Penn State College of Medicine Genome Sciences Facility provides consultation, instrumentation and services to both Penn State and non-Penn State investigators in genomic, epigenomic and transcriptomic studies.
The variety of instrumentation allows for capabilities ranging from highly focused analysis of candidate SNPs and mRNAs to whole genome, exome, epigenome and transcriptome sequencing. Services are also available for a variety of study designs extending from a few laboratory samples to large clinical projects involving hundreds or thousands of samples. The full bioinformatics service is also available for data analysis.
The facility receives either tissue, DNA/RNA or customer-generated NGS libraries. Samples are processed based on agreement reached during consultations on the design of the experiment. The facility develops new applications to accommodate state-of-the-art NGS technologies. Facility personnel conduct sequence-read alignment, secondary analysis (quantitation, variant calling, functional annotation, visualization, etc.) and follow-up with the interpretation of the results. The facility also provides support for grant writing and hands-on training for students and postdocs in NGS processing.
The facility resides in 5,000 square feet of newly renovated space, encompassing separate “pre-amplification” and “post-amplification” rooms to prevent any contamination of PCR-amplified materials to pre-processed input DNA/RNA samples. Four well-experienced staff members are available for assisting in project operations. In addition, the lab space is available for investigators who need temporary room for sample preparation.
All projects, submissions and pricing checks must be done through iLab.
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Instrumentation and Services
A variety of instrumentation is available in the Genome Sciences Facility for sample processing, sample quantitation and quality control, qPCR/digital PCR, microarray analyses, next-generation sequencing and other needs.
- QIAsymphony DNA/RNA Extraction Robot
- Bullet Blender (high-throughput tissue homogenizer)
- Covaris Adaptive Fcused Acoustics Ultrasonicator E-Series
Sample Quantitation and Quality Control
- Agilent 2100 Bioanalyzer
- Nanodrop ND-1000 Spectrophotometer
- Qubit Fluorometer
- Molecular Devices UV/Vis/Fluor Spectrophotometer (available in Drug Discovery Core)
- ABI QuantStudio 12K Flex system with available OpenArray block
- ABI QuantStudio 3D Digital PCR System
Next-Generation Sequencing (NGS)
- Agilent Genespring
- Illumina GenomeStudio
- Illumina iCompute
- Ingenuity Pathway Analysis
Additionally the facility contains refrigerators, -20 degrees C and -80 degrees C freezers, and associated small equipment (e.g., multichannel, electronic and accordion pipettes, centrifuges, etc.).
Nucleic Acid Isolation
Nucleic acid isolation is 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.
- 200 ul Whole Blood DNA prep
- 400 ul Whole Blood DNA prep
- 1000 ul Whole Blood DNA prep
- 200 ul DNA prep buffy coat
- 400 ul DNA prep buffy coat
- 400 ul RNA prep
- 800 ul RNA prep
- 1000 ul Saliva DNA prep
Covaris Adaptive Focused Acoustics Ultrasonicator
This equipment, providing DNA and chromatin shearing capabilities, is available in room C2706. The Covaris enables shearing of samples without thermal damage. The equipment is available on a sign-up basis.
Other options for sample extraction of RNA/DNA may be found in iLab.
Genome Sciences has Illumina sequencing instrumentation – MiSeq and HiSeq 2500 (retiring in the 2019-2020 fiscal year) and NovaSeq6000 – for both focused and large-scale sequencing by synthesis. Specific library construction services are available. Investigators who have a sequencing project should initiate the project through iLab. to receive a project ID before submitting samples.
For library construction, the facility recommends Illumina library preparation kits, and most other library preparation kits with Illumina adaptors will work with the MiSeq.
Both rapid-run and high-output workflows are now available on the HiSeq2500, which is retiring in the 2019-2020 fiscal year.
This offers scalable throughput and flexibility for virtually any sequencing method, genome and scale of project.
The Genome Sciences Facility provides a variety of library preparation services including whole genome, whole exome, ChIP-seq, Methylation-seq, RNA-seq and targeted resequencing. Listed below are currently operated services, but the facility is expanding the service to meet the needs of each investigator.
- Whole-genome sequencing library prep
- Whole-exome sequencing (Human) (minimum input DNA 1 ng for intact DNA, under R and D for FFPE DNA)
- Exome (other species, mouse, bovine, zebrafish)
- ChIP-sequencing library prep (minimum input DNA 0.5 ng)
- Low-input ChIP-seq library prep (minimum input DNA 0.05 ng)
- Methylation sequencing -ERRBS (enhanced reduced representation of bisulfite sequencing) library prep
- PolyA RNA seq library prep (strand-specific, minimum input RNA 50 ng)
- Total RNA seq library prep (rRNA depleted, human, mouse, rat, standard rRNA-depletion, strand-specific, minimum input RNA 100 ng)
- Low-input RNA-seq library prep (minimum input RNA 10 pg, or single cell)
- Low-input cDNA synthesis (minimum input RNA 10 pg, or single cell)
- Degraded low-input RNA-seq library prep (minimum input RNA 10 ng)
- Degraded low-input cDNA synthesis
- Small RNA seq library prep (minimum input RNA 1 ug)
- Low-input small RNA-seq library prep (minimum input RNA 100 ng)
- Single-cell RNA-seq (up to 96 samples)
A QC bioanalyzer run will be added (usually three runs with up to 11 samples per chip).
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 in the Genome Sciences facility are dual-labeled probe chemistry (TAQMAN) and SYBR green chemistry.
The facility’s recommendation for ease of use is to utilize the pre-optimized Primer/Probe Applied Biosystems Gene Expression Assays (Assays-on-Demand). These may be ordered through the facility.
The facility can assist in finding other sources should the desired gene not be available at ABI, either with another company or with design assistance.
QuantStudio 12KFlex with 384-well and available OpenArray block is in C2705.
384-well barcoded plates and seals may be purchased in the Supply Center.
Investigators may still use SDS software to create the plate and export the setup file into the “Incoming 7900 Plates” folder on the network drive under Research -> CoreFacility -> Results -> Genome Sciences Incoming. Go to File and choose “Export setup;” don’t send the SDS file itself.
QuantStudio licenses are also available for purchase through the core.
Analysis for Comparative Ct plates (RQ) can be done in the individual investigator’s lab using free ExpressionSuite software from LifeTechnologies.
For those using an unavailable species, or a gene not yet listed, the facility is 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 RealtimePrimers.com.
Investigators who wish to design their own primer/probe sets can use Primer Express, which is available in the facility (Room C2705).
A good source of information describing techniques and including troubleshooting may be found in the 2006 GenomeTech Real-Time PCR Tech Guide, which can be found in Box (Penn State Access ID login required).
Taqman Dual-Labeled Probe (5′-nuclease)
This is an oligo that is complementary to a portion of the gene of interest sequence between 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 the 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 the 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 on each dual-labeled probe. The disadvantage of using dual-labeled probes is that one dual-labeled probe must be purchased for each gene of interest.
This is a dye that will bind to double-stranded nucleic acid. As primers anneal and the polymerase extends to make new copies of the gene of interest, the amount of SYBR green increases proportionately. The rate at which new copies of the gene of interest are generated is inferred by the rate at which SYBR green fluorescence increases.
For details on any qPCR information, call 717-531-5823 or email Genome Sciences at firstname.lastname@example.org.
Penn State College of Medicine has partnered with Genewiz for Sanger DNA sequencing services.
- Register for a Genewiz account with the investigator’s lab’s specific Genewiz account ID.
- Once logged in to Genewiz, select “Create a Sequencing Order” from the upper left quadrant of the account home page.
- Select the sequencing order options that best meet project requirements.
- Enter all order information into the online order form or Excel order form.
- Once the Sanger DNA sequencing order is complete, print the order receipt, affix labeled samples to order receipt and place into a Ziploc bag for sample submission.
- Place the Ziploc bag containing the order receipt and samples in the Genewiz Drop Box at Penn State College of Medicine in Room C2705 by 4 p.m. weekdays. (Complete the sample log when dropping off samples at each Genewiz Drop Box to ensure effective submission.)
- Sequencing results will be accessible within the Genewiz account by 5 p.m. the business day following sample submission.
Genewiz Technical Support
Other DNA Sequencing Services
For additional questions regarding DNA sequencing, contact Yuka Imamura Kawasawa at email@example.com.
The Genome Sciences facility uses the Illumina platform for DNA methylation and genotyping arrays.
For focused genotyping/CNV/Methylation studies, the Infinium or GoldenGate process using the iScan instrument is extremely powerful. This platform allows investigators to choose a wide variety for specific research aims.
An important guideline for any microarray project is to use a minimum of three biological replicates per experimental (or control) group.
Investigators will receive a GenomeStudio project for methylation and other genotyping projects.
The Department of Public Health Sciences is also available for assistance in bioinformatics related to microarray analysis.
Ingenuity Pathway Analysis software is available for the analysis of genomic, transcriptomic and proteomic data.
To obtain an IPA login with the University’s license, email ResearchComputing@pennstatehealth.psu.edu with full name, Penn State email address, department and PI information.
Currently, access is free, and the software may be accessed from any location with an internet connection.
Procedures, Protocols and Forms
All RNA (total or mRNA/poly-A+) must be assessed for quality 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 helps to ensure quality and validity to future experiments.
At least 3 µl of RNA at a concentration greater than 0.1-0.5 µg/µl must be provided in a well-labeled tube for the quality control analysis.
RNA QC for up to 12 samples can be analyzed on the RNA 6000 Nano chip, and up to 11 samples can be analyzed on the RNA 6000 Pico chip (rare or micro-dissected samples).
These sample images were provided by Darran May, Aubree Hoover and Tom Sims as part of a presentation on the interpretation of Bioanalyzer 2100 RNA analysis.
Image of Perfect (Intact Eukaryotic) Total RNA
A: 5S subunit; prep-dependent.
B: There maybe a small peak present at approximately 24 seconds that represents 5s, 5.8s and tRNA. This is especially noted with phenol or Trizol extraction, and is eliminated when total RNA is prepped using Qaigen columns, which remove the small RNAs. (Substitute 16S and 23S for prokaryotic samples.)
C: Distinct 18s ribosomal subunit.
D: No small, well-defined peaks between ribosomal peaks.
E: Distinct 28S ribosomal subunit (usually approximately twice 18S).
F: Flat baseline throughout electropherogram.
Samples that result in the above electropherogram image are good to use for message RNA preparation.
Image of Partially Digested Total RNA
A: Intensities of the smaller degraded RNA increases.
B: Baseline between and to the left of the ribosomal peaks becomes jagged.
C: 18s ribosomal subunit.
D: Intensities of the peaks decrease.
E: 28S ribosomal subunit; in general, the 28S peak begins to degrade first.
F: Peak begins to shift toward the left of the electropherogram.
Samples that result in the above electropherogram image are borderline for message RNA preparation and should be under serious consideration of re-extraction.
Image of Severely Degraded Total RNA
A: Overall decrease in fluorescent signal.
B: Ribosomal peaks mostly degraded; generally 28S peak degrades first.
C: Baseline shifts from flat linke to distinct fragmented peaks.
D: Major shift of products toward left of electropherogram as smaller fragments are created by degradation.
Samples that result in the above electropherogram image should not be used to prep message RNA.
Image of Genomic-DNA-Contaminated Total RNA
A: Nano peak.
B: Genomic DNA skewing 8S peak.
C: Additional genomic DNA peak.
The sample may wrongly be considered degraded, and concentration may be inflated. Genomic DNA may interfere with downstream reactions if not removed. Treatment can be done during isolation with QIagen RNase-Free DNase or by post-isolation digestion.
(This sample was provided by Joan Tupper.)
For each sample, perform the following, then note the sample number and the nanodrop/bioanalyzer result (ng/µl).
- Add 1 ml Tri-Rgt to tissue; homogenize on ice. It is best to add 500 µl, homogenize, add the remaining 500 µl, and vortex well.
- Add 100 µl BCP; shake for 15 seconds. Let sit at room temperature for 15 minutes. Centrifuge for 16 minutes at maximum (10 to 13K) at 4 degrees C. Pipette top supernatant into new tube.
- Add 500 µl isopropanol; mix well. Let sit for 10 minutes at room temperature. Centrifuge for ine to 12 minutes at maximum (10 to 13K) at 4 to 25 degrees C. Pipette or dump liquid, leaving pellet.
- Add 1 ml 75 percent EtOH and vortex. Centrifuge six minutes at maximum (10 to 13K) at 4 to 25 degrees C. Dump EtOH, leaving pellet; air-dry for approximately eight minutes.
- Dissolve pellet in Rnase-free water (approximately 50 µl or less). Place on ice.
- Test by gel or bioanalyzer to ensure quality of RNA.
RNA quality control samples must be submitted by filling out the proper form (RNA Nano, Pico or DNA) and saving it to the Penn State College of Medicine network drive in the Research -> CoreFacility -> Results -> Genome Sciences Incoming -> ~QC Submissions folder.
Note that nano chips hold 12 samples, while pico chips hold 11.
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.
Investigators should use their name or other unique description and should ensure the tubes are labeled with something descriptive as well.
Completed results of all work are put in individual researcher folders inside the Research -> CoreFacility -> Results -> Genome Sciences folder.
Investigators should cite Penn State College of Medicine Genome Sciences and list the names of the instruments that were utilized either in the Materials and Methods section or the Acknowledgements section of any article.