Evaluation of susceptibility of pear and plum varieties and rootstocks to Ca. P. pyri and Ca. P. prunorum using Real-Time PCR

Real-time PCR was used to quantify phytoplasma concentration in fifty inoculated trees from five Prunus rootstocks and in forty-eight symptomatic pear and Japanese plum trees from orchards. Seasonal fluctuation of Ca. P. prunorum in different Prunus rootstocks, over three years, showed that the highest percentage detected by nested-PCR was in the ‘Garnem’ rootstock on nearly all sampling dates. Intra-varietal differences were also observed. Phytoplasma titer could be estimated by real time PCR in some trees of the rootstocks ‘Garnem’, ‘Barrier’, ‘GF-677’ and ‘Marianna’, and ranged from 4.7x105 to 3.18x109 phytoplasmas per gram of tissue. Quantification by real-time PCR was not possible in the ‘Cadaman’ trees analyzed, probably due to a lower phytoplasma titer in this variety. Samples from infected trees from commercial plots had different phytoplasma concentration and detection percentage depending on the variety, both being lower in ‘Fortune’ and ‘606’ Japanese plum and in ‘Blanquilla’ pear trees.

PCR detection of the pKM.19/ScrfI RFLP (D7S23), a marker closely linked to the cystic fibrosis mutation

Source: Description: pKM-19 is a 1.0 kb EcoRI human genomic fragment inserted in pUC13, that detects a Scrfl (CC/NGG) RFLP (1, 2). We report here the primer sequences suitable for the detection of this RFLP by PCR...

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Background: Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions. Methods: With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell. Results: The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation. Conclusion: The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy

Reproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages.

New methods for the concentration of viruses from urban sewage using quantitative PCR.

Viruses are among the most important pathogens present in water contaminated with feces or urine and represent a serious risk to human health. Four procedures for concentrating viruses from sewage have been compared in this work, three of which were developed in the present study. Viruses were quantified using PCR techniques. According to statistical analysis and the sensitivity to detect human adenoviruses (HAdV), JC polyomaviruses (JCPyV) and noroviruses genogroup II (NoV GGII): (i) a new procedure (elution and skimmed-milk flocculation procedure (ESMP)) based on the elution of the viruses with glycine-alkaline buffer followed by organic flocculation with skimmed-milk was found to be the most efficient method when compared to (ii) ultrafiltration and glycine-alkaline elution, (iii) a lyophilization-based method and (iv) ultracentrifugation and glycine-alkaline elution. Through the analysis of replicate sewage samples, ESMP showed reproducible results with a coefficient of variation (CV) of 16% for HAdV, 12% for JCPyV and 17% for NoV GGII. Using spiked samples, the viral recoveries were estimated at 30-95% for HAdV, 55-90% for JCPyV and 45-50% for NoV GGII. ESMP was validated in a field study using twelve 24-h composite sewage samples collected in an urban sewage treatment plant in the North of Spain that reported 100% positive samples with mean values of HAdV, JCPyV and NoV GGII similar to those observed in other studies. Although all of the methods compared in this work yield consistently high values of virus detection and recovery in urban sewage, some require expensive laboratory equipment. ESMP is an effective low-cost procedure which allows a large number of samples to be processed simultaneously and is easily standardizable for its performance in a routine laboratory working in water monitoring. Moreover, in the present study, a CV was applied and proposed as a parameter to evaluate and compare the methods for detecting viruses in sewage samples.

New methods for the concentration of viruses from urban sewage using quantitative PCR.

Viruses are among the most important pathogens present in water contaminated with feces or urine and represent a serious risk to human health. Four procedures for concentrating viruses from sewage have been compared in this work, three of which were developed in the present study. Viruses were quantified using PCR techniques. According to statistical analysis and the sensitivity to detect human adenoviruses (HAdV), JC polyomaviruses (JCPyV) and noroviruses genogroup II (NoV GGII): (i) a new procedure (elution and skimmed-milk flocculation procedure (ESMP)) based on the elution of the viruses with glycine-alkaline buffer followed by organic flocculation with skimmed-milk was found to be the most efficient method when compared to (ii) ultrafiltration and glycine-alkaline elution, (iii) a lyophilization-based method and (iv) ultracentrifugation and glycine-alkaline elution. Through the analysis of replicate sewage samples, ESMP showed reproducible results with a coefficient of variation (CV) of 16% for HAdV, 12% for JCPyV and 17% for NoV GGII. Using spiked samples, the viral recoveries were estimated at 30-95% for HAdV, 55-90% for JCPyV and 45-50% for NoV GGII. ESMP was validated in a field study using twelve 24-h composite sewage samples collected in an urban sewage treatment plant in the North of Spain that reported 100% positive samples with mean values of HAdV, JCPyV and NoV GGII similar to those observed in other studies. Although all of the methods compared in this work yield consistently high values of virus detection and recovery in urban sewage, some require expensive laboratory equipment. ESMP is an effective low-cost procedure which allows a large number of samples to be processed simultaneously and is easily standardizable for its performance in a routine laboratory working in water monitoring. Moreover, in the present study, a CV was applied and proposed as a parameter to evaluate and compare the methods for detecting viruses in sewage samples.