If you have pediatric translational research needs that are not listed here, please contact us as we are happy to discuss customizing capabilities to meet needs.

The capabilities of Translational research and Integrated Biology Lab (TRῙB) will continue to grow and expand to help meet the needs of the Department of Pediatric Scientific Investigators in the progression of their projects and in answering important scientific questions.

Consultation, Design and Budget

A meeting will be scheduled with the Principal Investigator (PI) and lab manager to discuss his or her research ideas, and to brainstorm the best approaches to investigating these ideas using the technologies available in the lab.  Our translational research coordinator/project manager is also available to help individual investigators and project teams design and facilitate a multi-disciplinary experimental design and determine what resources, including financial, are necessary/available to move projects forward.  Following the meeting, the best method to proceed forward with the research will be determined as well as a summary outlining the research goal(s), outcomes, and the scientific techniques/methods that will be utilized for the investigation.  In addition to this, a detailed budget will be developed summarizing the cost of all lab reagents, supplies, and hours spent working on the project.

TRῙB will need at least 2 weeks for consultation with PI, researching platforms/technologies/reagents, and co-designing strategy.

Sample Storage/Tracking

TRῙB can collect samples, process the samples per Principal Investigator (PI)/Investigators request, and aliquot the samples to avoid free/thaw cycles. Samples being collected by TRIB Scientists will be stored in TRῙB freezers located in the CCRC.  The CCRC/ TRῙB manages a Specimen Storage Facility dedicated to sample storage and tracking in accordance with International Society of Biologic and Environmental Repositories (ISBER) Best Practices and protocol defined sample parameters.  TRῙB is fully connected with the Indiana Biobank and can help identify/integrate samples from this data set as well.  A REDCap database will be used to inventory and track all specimens to be in accordance with ISBER.  REDCap was chosen for its convenient workflow and ability to integrate into existing REDCap databases (e.g. Indiana Biobank).  REDCap is currently used to track 81,000 projects, and has 771 active institutional partners from CTSA, GCRC, RCMI and other institutions located in 60 different countries.  Created by a Vanderbilt University informatics group, REDCap provides investigators with software that supplies support for data capture, survey creating, and data transfer and exportation into numerous statistical programs. 

Aliquoting

Samples for further analyses may be separated from one large volume into numerous vials of smaller volumes to avoid unnecessary freeze/thaw cycles during future use and for faster, easier distribution into subsequent experiments.  Low tension, filter pipet tips are used during the aliquoting process to ensure accuracy and minimal volume loss of the sample.

DNA/RNA Extraction/Isolation

DNA/RNA may be extracted and purified from whole blood, saliva, dried blood spots, buffy coat, serum, buccal swabs, and ffpe tissue slides.  This high-yield process is performed using QIAmp Midi and Maxi kits by Qiagen.  During this process, the cells are lysed and the cellular solution is placed onto a column. The DNA specifically binds to the silica-gel membrane of the column during centrifugation, while all other components and contaminants, such as PCR inhibitors and proteins, in the sample do not bind and thus flow through the column and are discarded during wash steps.  The purified DNA/RNA can then be detached from the column, collected in water or TE buffer, and is ready to use for future experimentation.  DNA/RNA extraction is an essential intermediary step required before proceeding to numerous future analyses.    

DNA or RNA Quantification

Precise quantification of DNA or RNA concentrations may be determined, in addition to obtaining 260/230 ratios to measure RNA purity.  Only minimal volumes of the precious samples are required for accurate readings using the Qubit Fluorometer System by Life Technologies, which utilizes fluorometric molecular dyes that emit their fluorescent signal only upon binding to specific target molecules.  These fluorescent probes can specifically bind to their target even when it is present in low concentrations.  Determining the 260/230 ratio of a sample identifies the purity of its RNA since nucleic acids absorb light at an absorbance of 260 nm, while many organic contaminants absorb light at 230 nm.  Therefore, the lower the 260/230 ratio, the higher the presence of contaminants found in the sample.  Having accurate concentrations of DNA/RNA samples is vital in completing further analyses just as the 260/230 ratios are important for effectiveness of gene expression and other experimentation.

Reverse Transcription

With the use of Qiagen’s QuantiTect Reverse Transcription kits, complementary DNA (cDNA) can be synthesized from any RNA template, including the more troublesome RNA templates that form complex secondary structures or have high GC content.  In addition, the cDNA is synthesized with complete elimination of the genomic DNA, which is essential for successful PCR probe design and future gene expression experimentation.  Qiagen’s kit proves high-yield cDNA even from low concentrations of the initial transcript for future use in gene expression and other experimentation.

Whole Genome Amplification (DNA)

Samples having low DNA concentrations (< 10 ng/µL) or low volumes may be amplified using Repli-G kits by Qiagen.  The amplification of these samples allows for “back up” DNA to be created for precious samples, which is especially important for samples that are in low supply and need to be used for experiments in the future.  The amplification can yield up to 10 µg of purified, stable DNA from small amounts of purified genomic DNA or directly from fresh or dried blood, buccal swabs, fresh or frozen tissue, or cells.  The resultant uniform DNA is ideal for PCR, restriction enzyme analysis, high-throughput applications, or any other subsequent experiment.

Proteomics

Proteomics is concerned with the structure and function of proteins.  The study of proteomics is more complex than that of genomics due to the impact that differences in translation and post-translational modifications have on gene expression.  The Bio-Rad Bio-Plex 3D Suspension Array System in particular is used to both detect and quantify multiple biomarkers or proteins (cytokines, chemokines, growth factors, etc.) present in a single sample simultaneously.  When using the Bio-Plex 3D, the multiplex immunoassay is performed on suspended magnetic beads coated with antibodies specific for a particular analyte, and can run up to 500 different beads (thus 500 different analytes) in a single assay.  In addition, the Bio-Plex 3D is more sensitive and has a larger dynamic range compared to the conventional ELISA.  As a result, in a matter of hours, the Bio-Plex is capable of analyzing up to 500 different analytes while requiring as little as 12.5 µL of sample.  

Genomics

Genomics focuses on the structure and the function of the genome.  The MiSeq sequencing system by Illumina is a personal sequencer used for target sequencing.  The MiSeq allows for the assembly of small genomes and target sequences using a terminator-based method which detects individual bases as they are added to the synthesized DNA strands, resulting in highly accurate base-by-base sequencing.  The QuantStudio 12 K-Flex Real Time PCR System by Life Technologies is designed for real-time PCR and can be used for SNP genotyping and gene expression.  The QuantStudio can increase throughput by up to 32-fold and can efficiently evaluate hundreds of genes of interest in thousands of samples during a single run.  

Coordination/Connection with Pharmacodynamic (PK/PD) Modeling Experts

Help with coordination of Pharmacokinetic-pharmacodynamic (PK/PD) models are mathematical expressions determined in relationship to the development of drugs.  PK/PD data is important in analyzing drug disposition and the relationships with absorption, distribution, metabolism, and excretion of specific drugs.  Techniques such as mass spectrometry are used to approximate the optimal dosage of drugs, therapeutic windows, and the mechanism employed by a given therapy.

Particle Sizing and Counting

The size, number, concentration, and mass of particles with various optical properties, densities, colors, and shapes can be determined using the Elzone II 5390 Particle Size Analyzer’s high resolution technology.  The Elzone is capable of performing counts and determining the size of very low concentration samples suspended in either an organic or inorganic electrolyte.  Particles as small as 0.4 µm in diameter can be analyzed.  To perform an analysis, the particles of interest are suspended in an electrolyte solution, and then are drawn, one particle at a time, through an opening called the Electrical Sensing Zone.  As particles cross through this zone, a voltage is applied, causing a volume displacement of the electrolytic solution which results in a voltage pulse.  This voltage pulse is proportional to the particle’s volume.  Particle counts are collected as the number of cells per milliliter.