Quantitative PCR Procedure Research Articles

Dynamic changes in Borrelia burgdorferi populations in Ixodes scapularis (Acari: Ixodidae) during transmission: studies at the mRNA level.

Many B. burgdorferi genes are regulated at the level of transcription during B. burgdorferi passage from ticks to mammals. Particular spirochete outer surface proteins of interest are OspA, OspC, and vlsE. The messenger RNA (mRNA) levels produced by these three genes were determined by a quantitative reverse transcription PCR (q-RT-PCR) procedure for spirochete populations in nymphal I. scapularis midguts and salivary glands at specific intervals during the feeding process. The mRNA values were compared to that of a standard, the mRNA levels of the constitutively expressed Flagellin (fla) gene. The levels of OspA and vlsE did not increase markedly in the midgut during feeding, but the mRNA levels of OspC increased significantly during feeding. In tick salivary glands, OspA mRNA levels actually decreased during feeding, while OspC levels increased six orders of magnitude. The mRNA levels of vlsE in tick salivary glands increased significantly only during the last 2 days of tick feeding. Overall, OspA mRNA was more abundant in tick midguts, whereas OspC and vlsE mRNA was more abundant in tick salivary glands. Further studies on the regulation of B. burgdorferi transcription activity during the act of transmission will lead to a better understanding of spirochete transmission dynamics, and hopefully facilitate the development of novel ways of interrupting the spread of Lyme disease.

Quantitative monitoring of the mRNA expression pattern of the TGF-β-isoforms (β1, β2, β3) during transdifferentiation of hepatic stellate cells using a newly developed real-time SYBR Green PCR

Current methods to determine the mRNA of the TGF-β-isoforms, β1, β2, and β3, are not sensitive enough to detect small alterations in the expression levels. Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-specific quantification is the possibility to be abstain from the need to compare results with a house-keeping gene having a different sequence and PCR efficiency. Reproducibility of the results and analytical variances of the real-time PCR assays were tested. In transdifferentiating rat hepatic stellate cells (HSC) the TGF-β1-mRNA was found to be the predominant isoform expressed followed by TGF-β3 and low amounts of TGF-β2-mRNA. An alteration of the TGF-β1,-β2, and -β3 ratio during HSC transdifferentiation could not be detected. Furthermore, the GAPDH mRNA expression varied during HSC activation, and thus is not recommended as a standard in real-time PCR quantifications.

Development of a real-time PCR procedure including an internal control for the measurement of HCMV viral load

Human cytomegalovirus (HCMV) infections are frequent in immuno-compromised patients. The recent development of real-time PCR procedures that allow the rapid quantification of genome load will be helpful for accurate monitoring of these infections. Two extraction procedures were evaluated using 30 blood samples that were processed pure and diluted (1/10). Repeatability and reproducibility of the quantitative PCR procedure using an internal control for amplification were analysed, and its sensitivity compared to a qualitative PCR procedure using 50 HCMV culture positive blood samples. The real-time PCR and qualitative PCR procedures were positive in 46 and 48 of the samples tested, respectively. Discrepancies were observed for samples with a low viral load. The sensitivity of the real-time PCR procedure was evaluated at 500 HCMV DNA copies per ml of sera. The use of an internal control concomitantly processed during the HCMV quantification did not alter the sensitivity of the procedure, and was relevant for the detection of putative PCR inhibitors that may interfere with the amplification process. This procedure was used to measure genome load in two bone marrow transplant patients with HCMV disease, confirming that this new PCR procedure should be used widely for diagnosing and monitoring HCMV infections in transplant patients.

Quantitative reverse transcriptase PCR to gauge increased protease-activated receptor 1 (PAR-1) mRNA copy numbers in the Wobbler mutant mouse.

Thrombin acts on cells through the surface protease-activated receptor 1 (PAR-1), a G-protein-coupled member of the seven-transmembrane domain superfamily. On neural cells, thrombin has deleterious effects, killing neurons through apoptosis. Consequently, knowledge of PAR-1 expression in the nervous system may help to elucidate the role of thrombin in neurodegenerative disease. We developed a mimic construct to facilitate the highly sensitive technique of quantitative reverse transcriptase to PCR (qRT-PCR) to measure the differential expression of low copy number PAR-1 mRNA in neurodegenerative model systems. In this article, we report our results comparing homozygous wobbler (wr/wr) mice and normal littermates. By optimizing the transcription and quantitative PCR procedures to facilitate rapid copy number determination in small RNA samples, we documented a fivefold greater level of PAR-1 mRNA in the cervical spinal cord of wr/wr.

Differential occurrence of mutations in mitochondrial DNA of human skeletal muscle during aging

Seven mtDNA mutations (five base substitutions and two deletions) were studied in skeletal muscle samples of 18 human subjects aged 1 hr to 90 years. Quantitative PCR procedures were applied to determine the incidence (frequency of occurrence) and abundance (percentage of mutant mtDNA out of total mtDNA). The base substitutions, in general, showed a very early onset, three such mutations being detectable in the muscles of infants aged 1 hr and 5 weeks. Of two disease-associated point mutations studied, 3243 A→G showed significant accumulation with age (P < 0.05), while 8993 T→G showed no significant age accumulation (P > 0.1). Moreover, three arbitrarily chosen mutations (not disease-associated) showed no age-associated accumulation: two (7029 C→T and 7920 A→G) showed little change over the years (P > 0.1), while the other (13167 A→G) showed a significant decrease (P < 0.05). Both the 4,977-bp and 7,436-bp deletions showed a significant age-associated occurrence (P < 0.01 and P < 0.05, respectively). The age of onset of detectable deletions is about 20–40 years; thereafter, the incidence and abundance of deletions tend to increase as a function of advancing age. The seven specific mutations were found to occur independent of each other, indicating the random nature of mtDNA mutations in skeletal muscle. Moreover, the age-associated accumulation of multiple deletions was observed in the same set of muscle tissues, each extract displaying a unique set of multiple PCR products. Thus, mutations in mtDNA occur differentially in human skeletal muscle during aging. Hum Mutat 11:360–371, 1998. © 1998 Wiley-Liss, Inc.

Differential occurrence of mutations in mitochondrial DNA of human skeletal muscle during aging.

Seven mtDNA mutations (five base substitutions and two deletions) were studied in skeletal muscle samples of 18 human subjects aged 1 hr to 90 years. Quantitative PCR procedures were applied to determine the incidence (frequency of occurrence) and abundance (percentage of mutant mtDNA out of total mtDNA). The base substitutions, in general, showed a very early onset, three such mutations being detectable in the muscles of infants aged 1 hr and 5 weeks. Of two disease-associated point mutations studied, 3243 A-->G showed significant accumulation with age (P < 0.05), while 8993 T-->G showed no significant age accumulation (P > 0.1). Moreover, three arbitrarily chosen mutations (not disease-associated) showed no age-associated accumulation: two (7029 C-->T and 7920 A-->G) showed little change over the years (P > 0.1), while the other (13167 A-->G) showed a significant decrease (P < 0.05). both the 4,977-bp and 7,436-bp deletions showed a significant age-associated occurrence (P < 0.01 and P < 0.05, respectively). The age of onset of detectable deletions is about 20-40 years; thereafter, the incidence and abundance of deletions tend to increase as a function of advancing age. The seven specific mutations were found to occur independent of each other, indicating the random nature of mtDNA mutations in skeletal muscle. Moreover, the age-associated accumulation of multiple deletions was observed in the same set of muscle tissues, each extract displaying a unique set of multiple PCR products. Thus, mutations in mtDNA occur differentially in human skeletal muscle during aging.

Myocardial angiotensin receptor type 1 gene expression in a rat model of cardiac volume overload

To investigate the regulation of the angiotensin receptor type 1 (AT1) in different organs in cardiac volume overload, we measured AT1 mRNA content in the atria, left and right ventricle, kidney and liver of rats with an aortocaval shunt, produced by infrarenal aortocaval puncture 4 weeks earlier. For angiotensin receptor mRNA quantitation a novel quantitative PCR procedure based on liquid phase hybridization was used that allowed the determination of absolute AT1 mRNA copy numbers and its comparison in different organs. Glyceraldehydephosphate dehydrogenase (GAPDH) mRNA was measured by RT-PCR to control externally equal mRNA content and quality of RNA extraction in shunt animals and controls. Heart weight was increased in the shunt animals, with the greatest increase in the atria. Blood pressure, plasma renin activity, plasma angiotensin I and II and aldosterone concentrations were not significantly altered. The AT1 mRNA content was significantly increased in the atria (shunt: 1167 +/- 350 copies AT1 mRNA/ng RNA vs controls: 803 +/- 240; p < 0.05). No change was found in the right or left ventricle, in the kidney and liver. The findings document that atrial hypertrophy in cardiac volume overload parallel with a significant increase in atrial AT1 mRNA content.

Comparison of Different Quantitative PCR Procedures in the Analysis of the 4977-bp Deletion in Human Mitochondrial DNA

Four quantitative PCR procedures were compared for analysing the level of the 4977 bp deletion in human mitochondrial DNA. A single preparation of total cellular DNA from the heart of a 69-year-old female subject was used for all four methods. We estimated the deletion to represent 0.005% of total mtDNA molecules using the serial dilution procedure and two internal standard methods with two separate sets of reference recombinant plasmids. However, the value obtained with the kinetic PCR analysis was 0.5%, two orders of magnitude higher. The internal standard procedures are likely to be the most accurate among the four methods used, but the more technically convenient serial dilution method would also be an appropriate choice.

Changes in expression of lymphocyte amyloid precursor protein mRNA isoforms in normal aging and Alzheimer's disease

We measured, by employing a quantitative reverse-transcriptase PCR procedure, the relative (to beta-actin) levels of amyloid precursor protein APP751 and APP770 mRNA isoforms in lymphocytes obtained from 64 cognitively intact subjects ranging in ages from 20 to 91 years and in 19 patients with sporadic Alzheimer's disease. A positive correlation was observed between the relative lymphocyte APP751 mRNA levels and subject age for the cognitively intact cohort. No difference in lymphocyte APP751 mRNA levels was observed between Alzheimer's disease patients and their age-matched controls (> 55 years of age). However, the ratio of lymphocyte APP751:APP770 mRNA levels was significantly lower in Alzheimer's disease subjects compared to the > 55-year-old cohort. This decreased ratio is most likely due to an average 31% increase in the lymphocyte APP770 isoform in Alzheimer's disease patients compared to 12% in the > 55-year-old cognitively intact group. Marked individual differences in amount of APP mRNA isoforms were encountered among all the subject groups and in the < or = 55-year-old cohort, a 10-fold variation in individual APP751 mRNA levels was observed. The relevance of these findings in lymphocytes to the pathogenesis of Alzheimer's disease is discussed.