Source: UNIVERSITY OF TEXAS AT TYLER, THE submitted to NRP
PURCHASE OF A HIGH THROUGHPUT RT-PCR MACHINE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1002033
Grant No.
2014-67014-21773
Cumulative Award Amt.
$49,265.00
Proposal No.
2013-02606
Multistate No.
(N/A)
Project Start Date
Feb 1, 2014
Project End Date
Jan 31, 2015
Grant Year
2014
Program Code
[A1111]- Plant Health and Production and Plant Products: Insects and Nematodes
Recipient Organization
UNIVERSITY OF TEXAS AT TYLER, THE
3900 UNIVERSITY BLVD
TYLER,TX 757990001
Performing Department
College of Arts & Sciences
Non Technical Summary
It is becoming increasingly clear that differences among individuals of the species and of different species arise largely from differences in the expression patterns of genes. For example, although humans and chimpanzees share many of the same genes, the differences between these two species are stark and clear. Studies have shown such stark differences can best be explained by differences in gene expression patterns. The proposed equipment will be used to investigate gene expression differences in a variety of animal and plant systems to understand both fundamental processes as well as phenomena that can be exploited for pest management and food production.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110104050%
2157299113050%
Goals / Objectives
This proposal is a request for the purchase of a high throughput quantitative, real time PCR (qRT-PCR) system that will enable multiple users in the Department of Biology, The University of Texas at Tyler, to undertake gene expression analyses on thousands of genes simultaneously. Specifically, we propose to purchase a fully integrated CFX-384 Real-Time PCR Detection System with CFX Automation System (Bio-Rad) priced at $98,530 of which we request 50% ($49,265) from USDA; non-federal matching funds are 50%. The equipment will enhance research infrastructure at UT Tyler and enable Biology faculty to generate preliminary data more efficiently and compete for grant funds more effectively.
Project Methods
Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously to identify gene regulatory networks. High throughput sequencing facilitates the assay of hundreds or thousands of genes simultaneously. The detection chemistry of qRT-PCR procedures is based on monitoring amplification products through either binding of dsDNA using ethidium bromide or SYBR green or target specific hybridization to single stranded DNA using molecular beacons or hydrolysis probe assays. Until recently, throughput has been limited to 384 reactions per run for individual laboratories (e.g., LightCycler 480 (Roche), ABI PRISM 7900 HT system (Applied Biosystems); CFX PCR Detection System expands the capacity to 7680 samples. Each system has its advantages and disadvantages but they all perform equally well in terms of sensitivity and robustness of the results. They vary in sample capacity, reagent use, and time required to complete the analysis.The CFX PCR detection system and the CFX automation system are equipped with a plate handler and a capacity to load twenty 384-well plates for a total of 7680 samples at a time which facilitates workflow automation, generation of large volume of data, and rapid analysis of the data with built-in software. The software enables the investigator to define PCR protocols for multiple experiments at once allowing for different protocols for each plate. This is a useful feature because the optimal PCR conditions are often gene-specific. The qRT-PCR software for the CFX system includes various components that allow the investigator to meet the MIQE guidelines for publishing qRT-PCR data. Finally, the system also facilitates stand-alone operation or operation with multiple plates very easily.

Progress 02/01/14 to 01/31/15

Outputs
Target Audience: The target audience for this project during the grant period included various faculty, graduate students, and undergraduate students in the Department of Biology at The University of Texas at Tyler. Changes/Problems: As mentioned, the major change is as follows, which was implemented with permission from the responsible Program Officer. After obtaining permission from the Program Officer in charge of this grant (Dr. Mary Purcell) in April 2014, the major objective of this project was modified slightly to purchase a more advanced PCR machine with no increase in cost. The machine that was purchased was Droplet Digital PCR Machine from BioRad (http://www.bio-rad.com/en-us/product/digital-pcr-technology/qx100-droplet-digital-pcr-system). What opportunities for training and professional development has the project provided? The opportunities for training with the purchase of the Droplet Digital PCR system are many and varied. Faculty undertaking gene expression and population genetic studies will be able to use the system to estimate the absolute abundances of alternate transcripts or alleles. Multiple graduate and undergraduate students, including underrepresented minorities and women, will be training in the state-of-the-art instrumentation, which will help them to reach their educational and career goals. How have the results been disseminated to communities of interest? Projects are currently underway and no products are available. When the projects are completed, the findings will be presented at professional meetings and published in peer-reviewed journals. The findings will also be disseminated to the general public through press releases. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? After obtaining permission from the Program Officer in charge of this grant (Dr. Mary Purcell) in April 2014, the major objective of this project was modified slightly to purchase a more advanced PCR machine with no increase in cost. The machine that was purchased was Droplet Digital PCR Machine from BioRad (http://www.bio-rad.com/en-us/product/digital-pcr-technology/qx100-droplet-digital-pcr-system). In conventional RT-PCR, the results of gene expression differences are usually expressed as a relative fold difference. An absolute differences in gene expression is not calculated. The Droplet Digital PCR System, however, has the capability to provide an absolute count of different transcripts in a PCR reaction. Each 50 ul PCR is emulsified into ~20,000 droplets and PCR is performed on the template DNA in each droplet. If all 20,000 droplets contain the same template, it can be concluded that only one transcript/allele is being expressed. However, a more common scenario is that a majority of the droplets will contain the most common transcript and the remainder will contain one or more alternate, rare transcripts. The purchase was finalized in September 2014 and installed the same month. A representative from BioRad provided onsite training to faculty and graduate students. The system is now fully operational and functional.

Publications