Barcoding Tetrahymena: discriminating species and identifying unknowns using the cytochrome c oxidase subunit I (cox-1) barcode.

DNA barcoding using the mitochondrial cytochromecoxidase subunit I (cox-1) gene has recently gained popularity as a tool for species identification of a variety of taxa. The primary objective of our research was to explore the efficacy of using cox-1 barcoding for species identification within the genusTetrahymena. We first increased intraspecific sampling forTetrahymena canadensis, Tetrahymena hegewischi, Tetrahymena pyriformis, Tetrahymena rostrata, Tetrahymena thermophila, and Tetrahymena tropicalis. Increased sampling efforts show that intraspecific sequence divergence is typically less than 1%, though it may be more in some species. The barcoding also showed that some strains might be misidentified or mislabeled. We also used cox-1 barcodes to provide species identifications for 51 unidentified environmental isolates, with a success rate of 98%. Thus, cox-1 barcoding is an invaluable tool for protistologists, especially when used in conjunction with morphological studies.

Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification.

The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line's species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification.

Size-based separations as an important discriminator in development of proximity ligation assays for protein or organism detection.

Proximity ligation is a powerful technique to measure minute concentrations of target protein with high specificity, and it has been demonstrated to be effective on a wide variety of protein targets. The proximity ligation assay (PLA) technique is shown to be compromised by the amplification of a nonspecific fluorescent product that is not indicative of protein presence, which was previously unidentified in a published procedure. This result illuminates the complexity of designing the optimal PLA and the possibility of using a size-based separation to increase the reliability of PLAs in general. Nucleic acid controls were developed to optimize the assay, which led to a novel end-point detection method that exploits microchip electrophoresis to size the products. This method provides a greater ability to distinguish a between the target protein's signal and noise in a PLA. The utility of the PLA is demonstrated by the detection of human pathogenic Escherichia coli O157:H7 bacteria, a pathogen at the root of many recent life-threatening food poisoning outbreaks. The results of the PLA show a detection limit of 100 E. coli O157:H7 cells with minimal cross-reactivity with gram positive control Staphylococcus aureus bacteria. The advantages of miniaturizing this process are the 100-fold reduction in volume, greatly reducing reagent requirements, and doubling of the thermocycling speed via noncontact infrared heating. This work, consequently, adds to the understanding of background fluorescence in PLAs, provides a method for evaluating nonspecific amplification, and shows that a qualitative PCR response indicative of the presence protein can be achieved with PLA.

Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena.

The mitochondrial cytochrome-c oxidase subunit 1 (cox1) gene has been proposed as a DNA barcode to identify animal species. To test the applicability of the cox1 gene in identifying ciliates, 75 isolates of the genus Tetrahymena and three non-Tetrahymena ciliates that are close relatives of Tetrahymena, Colpidium campylum, Colpidium colpoda and Glaucoma chattoni, were selected. All tetrahymenines of unproblematic species could be identified to the species level using 689 bp of the cox1 sequence, with about 11 % interspecific sequence divergence. Intraspecific isolates of Tetrahymena borealis, Tetrahymena lwoffi, Tetrahymena patula and Tetrahymena thermophila could be identified by their cox1 sequences, showing <0.65 % intraspecific sequence divergence. In addition, isolates of these species were clustered together on a cox1 neighbour-joining (NJ) tree. However, strains identified as Tetrahymena pyriformis and Tetrahymena tropicalis showed high intraspecific sequence divergence values of 5.01 and 9.07 %, respectively, and did not cluster together on a cox1 NJ tree. This may indicate the presence of cryptic species. The mean interspecific sequence divergence of Tetrahymena was about 11 times greater than the mean intraspecific sequence divergence, and this increased to 58 times when all isolates of species with high intraspecific sequence divergence were excluded. This result is similar to DNA barcoding studies on animals, indicating that congeneric sequence divergences are an order of magnitude greater than conspecific sequence divergences. Our analysis also demonstrated low sequence divergences of <1.0 % between some isolates of T. pyriformis and Tetrahymena setosa on the one hand and some isolates of Tetrahymena furgasoni and T. lwoffi on the other, suggesting that the latter species in each pair is a junior synonym of the former. Overall, our study demonstrates the feasibility of using the mitochondrial cox1 gene as a taxonomic marker for 'barcoding' and identifying Tetrahymena species and some other ciliated protists.

Species identification in cell culture: a two-pronged molecular approach.

Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the "barcode region" by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts.

Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT).

Herein, we present data demonstrating that the application of initial cell culture enrichment could significantly improve mycoplasma testing methods based on the nucleic acid amplification technology (NAT) including a polymerase chain reaction (PCR)/microarray method. The results of the study using Vero cells demonstrated that this cell culture is able (1) to support efficient growth of mycoplasmas of primary interest, i.e., species found to be cell line contaminants, (2) to increase the sensitivity of NAT assay to the detection limits of the conventional broth/agar culture methods, and (3) to reduce the time required for mycoplasma testing fourfold in comparison with the conventional methods. Detection and identification of mycoplasmal agents were conducted using a modified PCR/microarray assay based on genetic differences among Mollicutes in the 16S-23S rRNA intergenic transcribed spacer (ITS). The application of nano-gold/silver enhancement technology instead of previously used fluorescent dyes significantly simplified the readout of microarray results and allowed us to avoid using expensive scanning equipment. This modification has the potential to expand the implementation of microarray techniques into laboratories involved in diagnostic testing of mycoplasma contamination in cell substrates and potentially in other biological and pharmaceutical products.

Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing.

We have completed sequencing the 16S-23S rRNA intergenic transcribed spacer (ITS) region of most known Mycoplasma , Acholeplasma , Ureaplasma , Mesoplasma , and Spiroplasma species. Analysis of the sequence data revealed a significant interspecies variability and low intraspecies polymorphism of the ITS region among Mollicutes . This finding enabled the application of a combined polymerase chain reaction-microarray technology for identifying Mollicutes species. The microarray included individual species-specific oligonucleotide probes for characterizing human Mollicutes species and other species known to be common cell line contaminants. Evaluation of the microarray was conducted using multiple, previously characterized, Mollicutes species. The microarray analysis of the samples used demonstrated a highly specific assay, which is capable of rapid and accurate discrimination among Mollicutes species.

Quality prediction of cell substrate using gene expression profiling.

Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product.

The External RNA Controls Consortium: a progress report.

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

Exogenous reference RNA for normalization of real-time quantitative PCR.

We have utilized an in vitro transcribed 3' mRNA fragment of the plant gene ribulose bisphosphate carboxylase (RuBisCO) as an exogenous standard for normalization of quantitative PCR data. Both K562 cells and primary erythroid CD34+ progenitor cells were treated with sodium butyrate and changes in gamma-globin mRNA levels were assayed using a previously published TaqMan probe and primer set, while RuBisCO levels were assayed by a SYBR Green detection assay. The data presented show that a correction to measured gamma-globin induction was necessary with both cell types. The correction for the CD34+ progenitor cells was a striking 95% increase, while that for the K562 cells was 44%. The use of an exogenous reference such as in vitro transcribed mRNA for the RuBisCO plant gene provides a robust and sample-independent method for the normalization of quantitative PCR data in bacterial and animal cells.

Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore.

Multiplex quantitative PCR based on novel design of fluorescent primers is described. Fluorogenic primers are labeled with a single fluorophore on a base close to the 3' end with no quencher required. A tail of 5-7 nt is added to the 5' end of the primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial fluorescence of the primers that increases up to 8-fold upon formation of the PCR product. The hairpin oligonucleotides (DeltaG from -1.6 to -5.8 kcal/mol) may be as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Multiple fluorogenic primers were designed by specialized software and used for real-time quantitation of c-myc and IL-4 cDNAs in the presence of reference genes such as beta-actin, GAPDH and 18S rRNA. Targets of 10-10(7) copies were detected with precision in PCR using FAM-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method was also used to detect single nucleotide polymorphism of the human retinal degeneration gene by allele-specific PCR with end-point detection using a fluorescent plate reader or a UV-transilluminator. We conclude that fluorogenic mono-labeled primers are an efficient and cost-effective alternative to FRET-labeled oligonucleotides.