Comparison of different methods for preparation and characterization of total RNA from cartilage samples to uncover osteoarthritis in vivo

<p>Abstract</p> <p>Background</p> <p>The isolation of intact RNA can be very difficult when tissues are used that contain many RNAses or that are hard to homogenize, e.g. cartilage samples. Additionally, cartilaginous tissues are characterized by a low cellularity and an abundance of extracellular matrix (ECM) molecules. But given the growing interest in understanding pathogenesis of degenerative diseases, e.g. osteoarthritis (OA) and rheumatoid arthritis (RA), studies have to consider expression pattern of cells in its natural environment.</p> <p>Findings</p> <p>We compared the current RNA isolation methods for the extraction of high-quality RNA of snap-frozen biopsies from limited amounts of hypocellular cartilaginous tissue. The focus of the study was to gather information about procedure-related differences in RNA quality and yield. Here, we describe two protocols, the phenol/chloroform-free filter-based method (RN<it>Aqueous</it>™ kit) and the combined protocol (TRIzol^®/RNeasy Mini™ kit), working in a reproducible and reliable manner.</p> <p>Conclusions</p> <p>We conclude that preparation, storage, homogenization, and quality control are altogether critical steps for in-depth analysis of differential gene expression, especially in hypocellular tissues with highly crosslinked ECM like cartilage.</p

Zoonotic Chlamydiaceae Species Associated with Trachoma, Nepal - Volume 19, Number 12—December 2013 - Emerging Infectious Diseases journal - CDC

Trachoma is the leading cause of preventable blindness. Commercial assays do not discriminate among all Chlamydiaceae species that might be involved in trachoma. We investigated whether a commercial Micro-ArrayTube could discriminate Chlamydiaceae species in DNA extracted directly from conjunctival samples from 101 trachoma patients in Nepal. To evaluate organism viability, we extracted RNA, reverse transcribed it, and subjected it to quantitative real-time PCR. We found that 71 (70.3%) villagers were infected. ArrayTube sensitivity was 91.7% and specificity was 100% compared with that of real-time PCR. Concordance between genotypes detected by microarray and ompA genotyping was 100%. Species distribution included 54 (76%) single infections with Chlamydia trachomatis, C. psittaci, C. suis, or C. pecorum, and 17 (24%) mixed infections that includied C. pneumoniae. Ocular infections were caused by 5 Chlamydiaceae species. Additional studies of trachoma pathogenesis involving Chlamydiaceae species other than C. trachomatis and their zoonotic origins are needed

Zoonotic Chlamydiaceae species associated with trachoma, Nepal.

Trachoma is the leading cause of preventable blindness. Commercial assays do not discriminate among all Chlamydiaceae species that might be involved in trachoma. We investigated whether a commercial Micro-ArrayTube could discriminate Chlamydiaceae species in DNA extracted directly from conjunctival samples from 101 trachoma patients in Nepal. To evaluate organism viability, we extracted RNA, reverse transcribed it, and subjected it to quantitative real-time PCR. We found that 71 (70.3%) villagers were infected. ArrayTube sensitivity was 91.7% and specificity was 100% compared with that of real-time PCR. Concordance between genotypes detected by microarray and ompA genotyping was 100%. Species distribution included 54 (76%) single infections with Chlamydia trachomatis, C. psittaci, C. suis, or C. pecorum, and 17 (24%) mixed infections that includied C. pneumoniae. Ocular infections were caused by 5 Chlamydiaceae species. Additional studies of trachoma pathogenesis involving Chlamydiaceae species other than C. trachomatis and their zoonotic origins are needed

Detection of atypical Chlamydiaceae in roe deer (Capreolus capreolus)

International audienceInvestigations on fecal samples, vaginal swabs and sera from roe deer (Capreolus capreolus) in south-western France led to the detection of a non-classified Chlamydiaceae strain. A total of 85 vaginal swabs were sampled from roe deer that had been captured in 2012 (n = 42) and 2013 (n =43). Using a Chlamydiaceae family-specific real-time PCR, only one vaginal swab out of the 42 samples done in 2012 tested positive and was subsequently identified as Chlamydia (C.) psittaci. In contrast, 6/43 vaginal swab samples were positive in 2013. Four of these positive samples came from a single group of roe deer, captured in the Fabas plain. Fecal samples from this group of 9 females were subsequently analyzed, with 6 of them testing positive with the Chlamydiaceae-specific PCR. All positive samples collected in 2013 were negative when re-tested with C. abortus-, C. pecorum- and C. suis-specific real-time PCR assays. Sera from this group of 9 females were analyzed with two immunoassays (recomLine and ELISA). Whereas intense positive reactions with C. pneumoniae antigens were observed for all sera when tested with the recomLine test, none was positive with the C. abortus specific ELISA test. Comparative sequence analysis of the 16S, 23S rRNA and ompA gene sequences from 3 animals, as well as the MLST analysis from 2 animals, showed that this roe deer group likely harbored the same bacterium related to members of the family Chlamydiaceae. Notably, the roe deer strain formed a separate entity different from the currently recognized chlamydial species, with C. trachomatis, C. suis and C. muridarum appearing as its closest relatives

Type and origin of samples included in this study.

<p>Type and origin of samples included in this study.</p

High Frequency of <i>Chlamydia trachomatis</i> Mixed Infections Detected by Microarray Assay in South American Samples

<div><p><i>Chlamydia trachomatis</i> is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the <i>omp</i>A gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of <i>C</i>. <i>trachomatis</i> infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for <i>C</i>. <i>trachomatis</i> genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 <i>omp</i>A genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F.</p></div

Characteristic of samples with mixed <i>omp</i>A genotype or conflicting results between genotyping methodologies.

<p>Characteristic of samples with mixed <i>omp</i>A genotype or conflicting results between genotyping methodologies.</p

<i>C</i>. <i>trachomatis</i> genotype distribution according to genotyping technique.

<p><i>C</i>. <i>trachomatis</i> genotype distribution according to genotyping technique.</p

Distribution of <i>C</i>. <i>trachomatis</i> genotypes according to type of sample.

<p>Distribution of <i>C</i>. <i>trachomatis</i> genotypes according to type of sample.</p