Reverse Transcription-competitive PCR Research Articles

Non-invasive diagnosis of acute rejection in kidney transplants with delayed graft function

Delayed graft function (DGF) often occurs in kidney transplants from deceased donors. We wanted to provide studies giving more accurate non-invasive tests for acute rejection (AR). Using real-time PCR, we examined the expression of cytolytic molecules such as perforin, granzyme B, and fas-ligand along with serpin proteinase inhibitor-9. We also measured the expression of FOXP3, a characteristic gene of T-regulatory cells known to be involved in AR. These studies were conducted on peripheral blood monocytes, urinary cells, and 48 surveillance kidney biopsies taken from a total of 35 patients with DGF. Of these patients, 20 had a histopathological diagnosis of AR, whereas other 28 had characteristics of acute tubular necrosis (ATN). Expression of cytolytic and apoptotic-associated genes in the biopsy tissue, peripheral blood leukocytes, and urinary cells was significantly higher in patients with AR than that in patients with ATN. Diagnostic parameters associated with FOXP3 gene expression were most accurate in peripheral blood leukocytes and urine cells with sensitivity, specificity, positive and negative predictive values, and accuracy between 94 and 100%. Our study shows that quantification of selected genes in peripheral blood leukocytes and urinary cells from renal transplant patients with DGF may provide a useful and accurate non-invasive diagnosis of AR.

A reverse-transcription competitive PCR assay based on chemiluminescence hybridization for detection and quantification of hepatitis C virus RNA

A reverse-transcription competitive PCR (RT-cPCR) combined with chemiluminescence hybridization was designed for the detection and quantitative determination of serum hepatitis C virus (HCV) RNA. The concentration of HCV RNA was calculated based on an external standard curve that was generated by coamplification of internal competitor and target sequences in serial dilutions. The detection limit of the chemiluminescence RT-cPCR was 100 copies/ml (94 IU/ml). Meanwhile, the linear range for quantitation extended from 850 copies/ml (795 IU/ml) to 4.95×10 7 copies/ml. The performance of the current assay for measuring circulating HCV levels from 26 anti-HCV-antibody positive patients was compared with that of branched-chain DNA (bDNA) and nested RT-PCR assays. Eighteen patients had HCV RNA levels that exceeded the quantitation limit by the chemiluminescence RT-cPCR, but only 11 patients were quantitation-positive by the bDNA. A significant correlation of the quantitation values was found between the chemiluminescence RT-cPCR and the bDNA ( R 2=0.8391). Among the eight patients with HCV RNA titers below the quantitation limit, four remained positive by the chemiluminescence cRT-PCR, demonstrating the results in agreement with those using the nested RT-PCR. Furthermore, good linearity was revealed for the HCV genotypes 1b, 2a, 2b in 3-order magnitude diluted serum samples. In conclusion, the proposed chemiluminescence RT-cPCR method can detect quantitatively HCV RNA as accurately as the bDNA method and has sensitivity as high as nested RT-PCR.

Microchip and capillary electrophoresis for quantitative analysis of hepatitis C virus based on RT-competitive PCR

A method to quantitatively perform reverse transcription-competitive PCR (RT-cPCR) of hepatitis C virus followed by both microchip and capillary electrophoretic separation and detection was described. In this method, HCV wild-type (WT) RNA extracted from serum was coretrotranscribed and coamplified with a constant amount of recombinant internal standard (IS) RNA which had the same primer binding region as the target RNA and was constructed by removing a centrally located 25-bp segment from the target template. A linear calibration curve was constructed by adding IS RNA at a constant concentration of 8000 copies μl −1 into a series of RNA target standards ranging from 400 to 10 6 copies μl −1. The amplified IS and target DNA were detected by both capillary and microchip electrophoresis via laser-induced fluorescence (LIF) using Cy5-labelled primer as the fluorescence probe. The method was further demonstrated for the quantitation of clinical patients with low, medium, and high viral titer and the results were found to be comparable to those determined by the commercial bDNA assay.

Expression of the Proopiomelanocortin Gene in the Pituitaries of Rats of the Hypertensive Strain NISAG

Expression of the proopiomelanocortin (POMC) gene in NISAG rats with inherited stress-induced arterial hypertension has been investigated by the reverse transcription–competitive PCR method. Normotensive rats of the inbred Wistar Albino Glaxo (WAG) strain have been used as control. The effect of 3 h immobilization stress has been examined. The content of POMC mRNA in pituitaries of stressed NISAG rats exceeds that in nonstressed rats of the same strain by a factor of 2.4. Rats of the WAG strain do not demonstrate a significant increase in the level of POMC mRNA. In addition, the basic transcription level of the POMC gene in NISAG rats is higher than in WAG ones.

Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells.

Tubulointerstitial nephritis is the main manifestation of acute renal damage caused by leptospirosis, but the mechanism remains unexplored. Patients infected with LEPTOSPIRA: shermani in Taiwan disclosed tubular dysfunction particularly in the medullary thick ascending limb of loop of Henle (mTAL), and the related renal damage seems to be underestimated. To elucidate the mechanism of tubular damage, outer membrane protein extract from LEPTOSPIRA: was administered to a model of cultured mTAL cells derived from normal mice. The addition of outer membrane protein extract from L. shermani to cultured mTAL cells induced a significant nuclear DNA binding of the NF-kappa B transcription factor by electrophoresis mobility shift assay. Forty-eight h after adding the outer membrane protein extract (0.2 microg/ml) to the cultured cells, the expression of inducible nitric oxide mRNA increased by 4.2-fold, monocyte chemoattractant protein-1 by 3-fold, and tumor necrosis factor-alpha by 2.4-fold when compared with untreated cells examined by reverse transcription competitive-PCR. Supernatant nitrite, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha protein levels also increased by 1.8-, 7.1-, and 5-fold, respectively. An antiserum raised against L. shermani largely prevented these effects. Outer membrane protein extract from L. bratislava induced fewer effects than L. shermani, and the avirulent nonpathogenic L. biflexa serovar patoc did not induce significant effects in the mTAL cells. In conclusion, L. shermani infection may cause mTAL cell damage and inflammation through the NF-kappa B-associated pathway. Findings of this study may be important in understanding the pathogenesis of tubulointerstitial nephritis caused by these organisms.

Ultrasensitive in-house reverse transcription-competitive PCR for quantitation of HIV-1 RNA in plasma

An ultrasensitive version of an ‘in-house’ reverse transcription-competitive polymerase chain reaction assay described previously for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma was developed. The increase in sensitivity from 400 to 50 HIV-1 RNA copies/ml was achieved by pelleting virus particles from 1.8 ml plasma by centrifugation prior to RNA extraction, modifying competitor DNA structure and amounts, and redesigning primers. Quantitation of HIV-1 RNA in 130 samples tested previously by the standard assay showed that the two procedures yield comparable results (mean absolute difference, 0.26±0.20 log) and that the ultrasensitive version detects HIV-1 RNA below the threshold of sensitivity of the standard method. The ultrasensitive ‘in-house assay’ and the reference QUANTIPLEX HIV-1 RNA 3.0 had the same sensitivity and gave equivalent results (mean absolute difference, 0.19±0.11 log), as shown by parallel blinded testing of 47 plasma samples. Titration experiments with reconstructed plasma samples allowed the determination of a dynamic range of 50–500 000 HIV-1 RNA copies/ml for the ‘in-house’ system. The interassay coefficient of variation for samples nominally containing 200, 4000 and 80 000 HIV-1 RNA copies/ml were 33.4, 22.9 and 38.2%, respectively. The performance, turnaround time, and cost-effectiveness of this system make it suitable for medium-scale clinical application.

Quantitative Analysis of Constitutive and Inducible CYPs mRNA Expression in the HepG2 Cell Line Using Reverse Transcription-Competitive PCR

Drug interactions which affect drug metabolism are of clinical importance. It is, however, difficult to estimate drug interactions in human from results obtained in animal experiments. In our previous study, we demonstrated that a combination of the HepG2 cell line and semiquantitative reverse transcription-PCR (RT-PCR) could be used to evaluate the degree of CYP3A mRNA induction by various drugs. Using an RT-competitive PCR (RT-cPCR) with β-actin as the standard in this study, the constitutive and rifampicin (RFP)-induced expression of CYP3A4, CYP2C9, CYP2E1, and CYP1A2 mRNA in the HepG2 cells could be quantitatively and reproducibly determined. 120 h-treatment of HepG2 cells with 50 μmol/l RFP induced maximally 8.4- and 6.0-fold the expression of CYP3A4 and CYP2C9 mRNA, respectively, in comparison with untreated cells. On the other hand, mRNA level in CYP2E1 and CYP1A2 was not significantly changed by 50 μmol/l RFP after 24 to 120 h. To our knowledge, we report for the first time quantitative profiles of CYPs mRNA in HepG2 cells. This study demonstrates the efficiency of a combination of HepG2 cells and RT-cPCR in the evaluation of CYPs mRNA-induction by drugs.

Relationship between mRNA Levels Quantified by Reverse Transcription-Competitive PCR and Metabolic Activity of CYP3A4 and CYP2E1 in Human Liver

Reverse transcription-competitive polymerase chain reaction is a powerful and sensitive tool for quantifying the absolute amount of mRNA. Using this method with β-actin as the standard, we measured the mRNA level of CYP3A and CYP2E1 isoforms in human livers. We also determined the metabolic activities for both CYP isoforms. The absolute amounts of CYP3A4 and CYP2E1 mRNA in 15 liver tissues ranged from 15 to 5127 and 15163 to 69289 copies/104 copies of β-actin (341- & 3.6-fold), respectively. The testosterone 6β-hydroxylation for CYP3A4 and chlorzoxazone 6-hydroxylation activity for CYP2E1 ranged from 30 to 505 pmol/mg/min (16-fold) and from 0.59 to 2.73 nmol/mg/ml (3.6-fold), respectively. The correlation between the mRNA level and activity of CYP3A4 was significant (r = 0.94), while there was no significant correlation for CYP2E1 (r = 0.04). In conclusion, we observed a significant correlation between enzyme activity and mRNA expression for CYP3A4 but not for CYP2E1. This fact indicates that CYP2E1, in addition to being less variable between individuals than CYP3A4, differs in its regulation mechanism.

Clinical Evaluation of an In-House Reverse Transcription-Competitive PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

An in-house reverse transcription (RT)-competitive PCR (RT-cPCR) for the quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma samples was developed and validated. The procedure involves (i) extraction of RNA with spin columns, (ii) ready-to-use bead-mediated RT, (iii) competitive PCR in a microtiter plate, (iv) agarose gel electrophoresis of the reaction products, and (v) densitometric analysis of the digitized image of the gel. Quadruplicate tests and dilution studies showed that the sensitivity and intertest coefficient of variability of the RT-cPCR are comparable to those of the reference AMPLICOR HIV-1 MONITOR test. The results obtained by the two assays with a panel of 45 clinical samples were in good agreement (mean difference, 0.36 +/- 0.25 log units). Analysis of 1,982 clinical samples by the in-house RT-cPCR yielded the typical range of plasma HIV-1 RNA levels with the expected inverse correlation between CD4 counts and HIV-1 RNA titers. In addition, testing of plasma from 36 subjects at weeks 0 and 4 with respect to the time of initiation of protease inhibitor therapy detected a significant decrease in HIV-1 viremia. The mean reduction in the HIV-1 RNA level was 0.914 log unit for those receiving saquinavir (P = 0.0210), 1.584 log units for those receiving indinavir (P = 0.0047), and 1.904 log units for those receiving ritonavir (P < 0.0001). The in-house RT-cPCR assay is simple to develop and perform and allows quantitation of HIV-1 RNA in 100 to 200 samples per operator per week. Since the cost is 1/8 to 1/10 of those of reference commercial assays, this procedure could be conveniently used in medium-scale laboratories.

The expression of ob gene is not acutely regulated by insulin and fasting in human abdominal subcutaneous adipose tissue.

The regulation of ob gene expression in abdominal subcutaneous adipose tissue was investigated using a reverse transcription-competitive PCR method to quantify the mRNA level of leptin. Leptin mRNA level was highly correlated with the body mass index of 26 subjects (12 lean, 7 non-insulin-dependent diabetic, and 7 obese patients). The effect of fasting on ob gene expression was investigated in 10 subjects maintained on a hypocaloric diet (1045 KJ/d) for 5 d. While their metabolic parameters significantly changed (decrease in insulinemia, glycemia, and resting metabolic rate and increase in plasma ketone bodies), the caloric restriction did not modify the leptin mRNA level in the adipose tissue. To verify whether insulin regulates ob gene expression, six lean subjects underwent a 3-h euglycemic hyperinsulinemic (846 +/- 138 pmol/liter) clamp. Leptin and Glut 4 mRNA levels were quantified in adipose tissue biopsies taken before and at the end of the clamp. Insulin infusion produced a significant threefold increase in Glut 4 mRNA while leptin mRNA was not affected. It is concluded that ob gene expression is not acutely regulated by insulin or by metabolic factors related to fasting in human abdominal subcutaneous adipose tissue.

Acute regulation by insulin of phosphatidylinositol-3-kinase, Rad, Glut 4, and lipoprotein lipase mRNA levels in human muscle.

We have investigated the acute regulation by insulin of the mRNA levels of nine genes involved in insulin action, in muscle biopsies obtained before and at the end of a 3-h euglycemic hyperinsulinemic clamp. Using reverse transcription-competitive PCR, we have measured the mRNAs encoding the two insulin receptor variants, the insulin receptor substrate-1, the p85alpha subunit of phosphatidylinositol-3-kinase, Ras associated to diabetes (Rad), the glucose transporter Glut 4, glycogen synthase, 6-phosphofructo-l-kinase, lipoprotein lipase, and the hormone-sensitive lipase. Insulin infusion induced a significant increase in the mRNA level of Glut 4 (+56 +/- 13%), Rad (+96 +/- 25%), the p85alpha subunit of phosphatidylinositol-3-kinase (+92 +/- 18%) and a decrease in the lipoprotein lipase mRNA level (-49 +/- 5%), while the abundance of the other mRNAs was unaffected. The relative expression of the two insulin receptor variants was not modified. These results demonstrate an acute coordinated regulation by insulin of the expression of genes coding key proteins involved in its action in human skeletal muscle and suggest that Rad and the p85alpha regulatory subunit of phosphatidylinositol-3-kinase can be added to the list of the genes controlled by insulin.