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J Biomol Tech. 2008 February; 19(1): 88–93.
PMCID: PMC2359592

RESEARCH GROUP ABSTRACTS

J Biomol Tech. 2008 February; 19(1): 88–93.

RG1 Molecular Interactions Research Group Session: (A) Discussion of Label-Free Technologies Survey and (B) Tutorial on Isothermal Titration Calorimetry

The number and types of label-free molecular interactions technologies is expanding at a rapid rate. There is a large diversity of these technologies in terms of the types of applications that they are best suited for, the cost of the instrumentation, the ease of software, the amounts of reagents needed to use them, and the technological expertise required to use them. The Molecular Interactions Research Group (MIRG) has conducted a survey to characterize this diversity and point to areas of need for future technologies. A roundtable discussion will be held to review the survey results. Additionally, a short tutorial will be given on one of the most rigorous label-free technologies, isothermal titration calorimetry (ITC). A key benefit of ITC is that it does not require immobilization to a surface.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG3 2008 Microarray Research Group (MARG Survey): Sensing the State of Microarray Technology

Over the past several years, there has been enormous growth and evolution in microarray technology and application. In its continued efforts to track this evolution and transformation, the ABRF-MARG has once again conducted a survey of international microarray facilities and individual microarray users. The goal of the survey is to profile the current state of microarrays and to gain insights into new trends in the field. The survey is composed of seven parts: General Section, Microarray Platforms, Service Options and Throughput, Commercial Platforms, Bioinformatics, Data Management, Next Generation Technologies, and Future Directions. Questions for each section addressed instrumentation, protocols, staffing, funding, and work flow in a microarray facility. This is the fifth microarray survey conducted by the ABRF-MARG; the last survey was conducted in 2005. The results of the survey are presented and new trends are discussed. Additionally, the survey was evaluated against past surveys to provide insights into the growth and evolution of the community of microarray researchers. This abstract does not reflect EPA policy.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG4 Inferring Protein Quantities from Peptides: Considerations in Selecting Peptide Sequences for Synthesis as Stable-Isotope-Labeled Standards

The proteomic standards research group (sPRG) is designing a study to quantify a subset of proteins within a complex mixture, such as human serum/plasma. In accordance with current mass spectrometry–based approaches, protein quantities are inferred from measurements of proteolytic peptides; typically, values determined from at least three peptides are averaged to improve precision. Absolute quantitation (in contrast to relative) requires reference standards of known concentration; e.g., stable-isotope-labeled synthetic peptides. We discuss the assumptions underpinning MS-based quantitative proteomics and present considerations in selecting peptide sequences to synthesize as stable-isotope-labeled standards. Among these considerations are (1) minimizing the impact of heterogeneous proteolytic cleavages, (2) avoiding sequences duplicated in other proteins, (3) avoiding unstable sequences prone to cleavage, deamidation, oxidation, and cyclization, (4) avoiding SNPs, alternative splice isoforms, and heterogeneous post-translational modifications, and (5) LC-MS “detectability” of individual sequences.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG5 Nucleic Acid Research Group 2007–2008 Study: A Comparison of Different Priming Strategies for cDNA Synthesis by Reverse Transcriptase as Measured by Real-Time RT-qPCR

Real-time reverse transcriptase quantitative PCR (RT-qPCR) has become well established as the method of choice by researchers to detect and quantify nucleic acid levels and is offered as a service in many shared resource facilities. As this technology continues to mature, the need to understand where sources of variation occur is critical to ascertaining best strategies for assay development. Recent publications have focused on the reverse transcription step1 and more specifically the random primer2 used in the generation of the cDNA as having an impact on assay sensitivity. While many laboratories use assay-specific primers for priming cDNA synthesis, the need to also utilize nonspecific priming strategies during the RT step is necessary if developing a multiplexed assay.

NARG has proposed a two-year study to determine the best priming strategies for cDNA synthesis by reverse transcriptase as measured by real-time RT-qPCR. The first year is an internal study within the Nucleic Acid Research Group comparing the ability of random primers of increasing length (6 to 21 bases in steps of 3 bases) in initiating cDNA synthesis. The set of random primers was compared to oligo-dT, anchored oligo-dT, combinations of random primer and oligo-dT, assay-specific primer, and no primer with and without reverse transcriptase. A high-temperature RNaseH-RT enzyme commonly used in this community (NARG Survey 2007), Superscript III, will be used as the reverse transcriptase enzyme. The goal of this study is to determine which single or combinatorial priming strategies are best suited for the most comprehensive synthesis of cDNA from total RNA for real-time RT-qPCR. The data from this study will be presented along with a discussion of the proposed Part II of the study, which will involve outside investigators interested in participating.

REFERENCES

  • Stahlberg A, Hakansson J, Xian X, Semb H, Kubista M. Properties of the reverse transcription reaction in mRNA quantification. Clinical Chemistry. 2004;50:509–515. [PubMed]
  • Stangegaard M, Dufva IH, Dufva M. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA. BioTechniques. 2006;40(5):649–657. [PubMed]
J Biomol Tech. 2008 February; 19(1): 88–93.

RG6-S NARG 2007–2008 Research Study: A Comparison of Different Priming Strategies for cDNA Synthesis by Reverse Transcriptase as Measured by Real-Time RT-qPCR

Real-time reverse transcriptase quantitative PCR (RT-qPCR) has become the method of choice to quantify transcript levels. Unfortunately, the ability to generate adequate quantities of cDNA for use with qPCR continues to challenge investigators. Recent publications1,2 focused on the reverse transcription step and more specifically the impact on assay sensitivity of the random primer used in the generation of the cDNA. While many laboratories use assay-specific primers for priming cDNA synthesis, the need to also utilize nonspecific priming strategies during the RT step has become paramount for some RT-qPCR assay designs (e.g., multiplexed assays). The Nucleic Acid Research Group (NARG) has conducted a study to evaluate priming strategies for generating cDNA for use with real-time RT-qPCR. The goal of this study is to determine which single or combinatorial priming strategies are best suited for the most comprehensive synthesis of cDNA from total RNA for real-time RT-qPCR. The study was designed to evaluate the ability of random primers of increasing length (6 to 21 bases in steps of 3 bases) in initiating cDNA synthesis. The set of random primers was compared to oligo-dT, anchored oligo-dT, combinations of random primer and oligo-dT, assay-specific primer, and no primer with and without reverse transcriptase. A high-temperature RNaseH-RT enzyme commonly used in this community (NARG Survey 2007), Superscript III was selected as the reverse transcriptase enzyme.

REFERENCES

  • Stahlberg A, Hakansson J, Xian X, Semb H, Kubista M. Properties of the reverse transcription reaction in mRNA quantification. Clinical Chemistry. 2004;50:509–515. [PubMed]
  • Stangegaard M, Dufva IH, Dufva M. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA. BioTechniques. 2006;40(5):649–657. [PubMed]
J Biomol Tech. 2008 February; 19(1): 88–93.

RG7 ESRG Study 2008: Investigation into Poly–Amino Acid N-Terminal Tags and Their Effects on Automated Edman Degradation

For decades, Edman degradation has been an invaluable tool for protein characterization. Though other techniques have surpassed Edman chemistry in ease, cost, and utility for routine protein characterization, automated protein sequencing remains the most effective tool for obtaining N-terminal amino acid sequence information. A common affinity tag for protein purification is a polyhistidine sequence, which may be conjugated to either termini of the protein. After expression, His-tagged proteins are readily purified via chelation with an immobilized metal affinity resin. Determining the N-terminal sequence for several amino acids beyond the His tag is important for confirming the proper expression of the protein. N-terminal His tags are often found to be problematic with regards to Edman sequencing, with poor repetitive yields and overall low signal. Therefore, the question is posed: is this the result of a homopolymeric amino acid sequence in general, or specifically the presence of histidine in the affinity tag sequence?

The Edman Sequencing Research Group (ESRG) of ABRF has enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. The laboratories were asked to sequence the same protein engineered in three configurations: (1) with an N-terminal poly-His tag, (2) an N-terminal poly-Ala tag, or (3) no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield information and the amount of lag were evaluated. Information on instrumentation and sample treatment were also collected.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG8-M ESRG Study 2008: Investigation into Homopolymeric Amino Acid N-Terminal Sequence Tags and their Effects on Automated Edman Degradation

For decades, Edman degradation has been an invaluable tool for protein characterization. Though other techniques have surpassed Edman chemistry in ease, cost, and utility for routine protein characterization, automated protein sequencing remains the most effective tool for obtaining N-terminal amino acid sequence information. A common affinity tag for protein purification is a polyhistidine sequence, which may be conjugated to either termini of the protein. After expression, His-tagged proteins are readily purified via chelation with an immobilized metal affinity resin. Determining the N-terminal sequence for several amino acids beyond the His tag is important for confirming the proper expression of the protein. N-terminal His tags are often found to be problematic with regards to Edman sequencing, with poor repetitive yields and overall low signal. Therefore, the question is posed: is this the result of a homopolymeric amino acid sequence in general, or specifically the presence of histidine in the affinity tag sequence? The Edman Sequencing Research Group (ESRG) of ABRF has enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. The laboratories were asked to sequence the same protein engineered in three configurations: (1) with an N-terminal poly-His tag, (2) an N-terminal poly-Ala tag, or (3) no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield information and the amount of lag were evaluated. Information on instrumentation and sample treatment were also collected.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG9-T Results from the 2008 DNA Sequencing Research Group Difficult Template Sequencing Study

In the past, the DNA Sequencing Research Group (DSRG) has conducted research studies on DNA templates that were moderately GC rich (DSRG 1997 study) or contained repeat elements (DSRG 2003 study). Though improvements in DNA sequencing chemistries have helped with difficult templates, one of the remaining challenges for classical Sanger DNA sequencing is the ability to effectively sequence through various types of difficult regions in DNA templates. In the 2008 DSRG difficult template sequencing study, we have expanded the number of templates and nature of the difficult sequences, and have designed the study to identify and develop optimal protocols for such templates.

The templates in this study include a moderate GC-rich region (up to 72%), a very GC-rich region (up to 95%), a long non-repeat di-nucleotide region, an Alurepeat, a 19-base-long G/C homopolymer, and a hairpin-containing template.

The same set of difficult templates (prep method/concentration/primer) were distributed to all willing participants and the study will be carried out in two phases:

Phase 1: Participants will apply a protocol of their choice, each condition will be performed in triplicate, and the data will be returned to the DSRG for analysis.

Phase 2: The DSRG will recommend optimal protocols for each template and participants will re-sequence all templates in triplicate utilizing the three best protocols selected from phase 1.

The DSRG has a long and successful track record of conducting inter-laboratory research studies to optimize protocols for DNA sequencing, providing benchmarking opportunities as well as resources for troubleshooting DNA sequencing reactions and instrumentation. Here, we present the results of the DSRG 2008 difficult template study, with the goal of defining optimal sequencing protocols for these types of difficult templates.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG10 Sequencing of Difficult DNA Templates: The 2008 DSRG Difficult Template Sequencing Study

The DNA Sequencing Research Group (DSRG) has a long and successful track record of conducting inter-laboratory research studies to optimize protocols for DNA sequencing, providing benchmarking opportunities and resources for troubleshooting DNA sequencing reactions and instrumentation. One of the remaining challenges for classical Sanger DNA sequencing is the ability to effectively sequence through various difficult regions in DNA templates. In the past we have conducted such studies on templates that were moderately GC rich (DSRG 1997 study) or contained repeat elements (DSRG 2003 study). Though improvements in DNA sequencing chemistries have helped with difficult templates, challenges still remain. In the 2008 DSRG difficult template sequencing study, we have expanded the number of templates and nature of the difficult sequences, and have designed the study to identify and develop optimal protocols for such templates.

The templates in this study include: (a) a moderate GC-rich region (up to 72%) and a very GC-rich region (up to 95%): (b) a long non-repeat di-nucleotide region; (c) an Alurepeat; (d) a 19-base-long G/C homopolymer, and (e) a hairpin-containing template.

The same set of difficult templates (prep method/concentration/primer) will be distributed to all willing participants and the study will be carried out in two phases:

Phase 1: Participants will apply a protocol of their choice, each condition will be performed in triplicate, and the data will be returned to the DSRG for analysis.

Phase 2: The DSRG will recommend optimal protocols for each template and participants will re-sequence all templates in triplicate utilizing the three best protocols selected from phase 1.

Results of the DSRG 2008 difficult template study will be presented, anticipating that this study will result in defining optimal sequencing protocols for these types of difficult templates.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG11 ABRF iPRG2008 Study: Assessing the Quality and Consistency of Protein Reporting on a Common Dataset

A significant challenge in proteome informatics is accurate and concise reporting of protein identification data that result from mass spectrometry–based proteomic workflows. The Paris Guidelines represent the proteomics community’s efforts to devise a standard methodology for reporting protein identification data. Conformance to these criteria is affected by the bioinformatics tools used, how one understands the issues, and interpretation of the reporting criteria. At ABRF2007 in Tampa, FL, the Bioinformatics Committee (BIC) of the ABRF sPRG presented a study assessing the consistency of protein reporting among a group of experts. In the present work, the newly formalized Proteome Informatics Research Group (iPRG) presents the results of a study that was designed to assess the quality and consistency of protein identification analysis across the broader ABRF community. The major goals of this study were to assess the present status of the field and to provide a benchmark for the quality of protein inference on a realistically complex dataset.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG12 Evaluation of Expression and Purification Strategies to Maximizing the Yield of a Recombinant Protein in E. coli.

The current study explores the use of different protein expression approaches to maximize the yields, quality and biological activity of a common recombinant protein, expressed in E. coli. This study will use a simple plasmid that expresses the yeast alcohol dehydrogenase (ADHI) protein in E. coli, with a C-terminus 6X-His tag to provide an easy means of purification. The participants will be asked to vary factors such as E. coli host strain, growth media, growth temperature, induction conditions, cell lysis buffers, and purification methods, to improve the final yield. The primary goal of this study is to document the breadth of approaches applied by the scientific community to this task and highlight the best method. The final comparative data on yield, purity, and activity will be presented at the 2008 ABRF meeting.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG13 What is Genotyping, and Why is the Definition Different for So Many Laboratories? Results from the FARG 2007 Survey

A large number of laboratories, both core and non-core based, are actively performing “genotyping.” However, the definition of genotyping may be different depending on the laboratory. More specifically, differing laboratories are performing genotyping and using genotyping technologies for drastically different research aims. Therefore, the implementation and measurement of the genotyping technologies’ performance have different important factors. To this end, members of the Fragment Analysis Research Group (FARG) have designed a survey to capture the differing types of genotyping technologies routinely implemented in laboratories, the research aims of said technologies, and, importantly, the demographics of such laboratories performing genotyping. The FARG 2007 genotyping survey was an online survey, available to a wide community of genotypers, including ABRF membership and Genomeweb, as well as numerous listserves, and represents a broad sampling of both the core and non-core laboratories performing genotyping. This presentation will focus on the collation of survey results, focusing on the different implementation of genotyping technologies. The main aim is to describe the similarity and outline differences of genotyping technologies and their specific implementation used to achieve described research aims. We will also examine the use of ancillary equipment implementation and breadth of analyses for each genotyping technology in accordance with these aims. Finally, we intend to summarize the different methods and draw inferences as to their potential selection, as a preliminary way to develop best practices, as they apply to the respondents and their use of individual genotyping technology.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG14-S Defining the Demographics of the Genotyping Laboratory: Results from the FARG 2007 Survey

A large number of laboratories, both core and non-core based, are actively performing “genotyping.” However, the definition of genotyping may be different depending on the laboratory. More specifically, differing laboratories are performing genotyping and using genotyping technologies for drastically different research aims. Therefore, the implementation and measurement of the genotyping technologies’ performance have different important factors. To this end, members of the Fragment Analysis Research Group (FARG) have designed a survey to capture the differing types of genotyping technologies routinely implemented in laboratories, the research aims of said technologies, and, importantly, the demographics of such laboratories performing genotyping. The survey was available to a wide community of genotypers, including ABRF membership and Genomeweb, as well as numerous listserves, and represents a broad sampling of both the core and non-core laboratories performing genotyping. This presentation will focus on the collation of survey results, focusing on the demographics of the genotyping laboratories that responded to the survey. Important differences in these demographics often help define genotyping and the scope of work. In addition, the physical size and methods of funding from each laboratory help shape the demographics and will be considered in such an analysis. Finally, we will examine the differences between core and non-core laboratories and provide areas of overlap as well as explain key differences.

J Biomol Tech. 2008 February; 19(1): 88–93.

RG15-M ABRF 2007 Survey: Service Laboratory Funding

The ABRF Survey Committee surveyed the ABRF membership and scientists at-large concerning the current state of funding in service-oriented laboratories. Questions were designed to elicit responses concerning service offerings, cost recovery, capital equipment funding, and future outlook. The Web-based survey, available for three weeks, achieved participation from 209 respondents in 13 countries, with 77% of the respondents representing academic laboratories. Most respondents (75%) directed laboratories.

Responses indicated that laboratories depend largely on institutional support and customer recharges to fund operations. NIH and NSF shared instrumentation grant programs were considered critical to meeting future needs. Breakdown of the sources supporting capital equipment acquisitions, operations, and lab director salary will be presented.

Acknowledgements: JTS—Funded by NCI Contract No. N01-CO-12400. The ABRF Survey Committee would also like to acknowledge the ABRF Membership Committee for critical reading and suggestions for the survey.


Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities