We progressively focused our study on Bcl xL anti apoptotic protein. Indeed, ribonuclease protection assay exposed that bcl xL displayed numerous ranges of mRNA expression in response to cisplatin amongst cell lines, whereas, as an illustration, no relationship could possibly be established concerning bcl expression and cellular response to cisplatin. Several data have recommended that Bcl loved ones might be differentially regulated in accordance on the tissue, and that some members could overcome the position of other folks in a tissue exact manner. Bcl xL, structural and practical analogue of Bcl , could so overcome the perform of Bcl in some cases . Bcl x gene is alternatively spliced into two different mRNAs, the very first 1 encoding for that anti apoptotic long kind of Bcl x , another 1 encoding to the professional apoptotic short kind of Bcl x , which appears like a dominant negative of Bcl and Bcl xL proteins . Like Bcl protein, Bcl xL has become localized in endoplasmic reticulum, nuclear membrane and external mitochondrial membrane , this latter localization currently being necessary for its intervention within the handle of mitochondrial apoptotic pathway.
In our examine, western blot and immunochemistry examination indicated that Bcl xL was expressed in every one of the examined ovarian cell lines and tumor samples . Immunocytochemistry showed that Bcl xL was localized within the cytoplasm, as expected. In addition, the patchy staining observed right after Bcl xL immunostaining, too as electron microscopy, specified Vemurafenib clinical trial that Bcl xL was largely located in mitochondria , as previously described by other people . In contrast, Bcl xS protein expression was undetectable in the many cases, which is not surprising according for the potent pro apoptotic function of this protein. The substantial percentage of Bcl xL expressing tumors is in agreement with the success of other research, by which this percentage varied from to . Our final results didn’t permit to correlate Bcl xL basal expression with sensitivity to cisplatin, considering that this protein was expressed in every one of the tumors and each of the cell lines, in spite of their big difference of response to treatment method.
Then again, the hyperlink involving basal expression of Bcl xL in tumors and patients’ survival has certainly not been obviously established, whether or not this expression more info here was shown to get predictive of a shorter disorder free of charge interval . This could be in component as a consequence of the higher proportion of tumors constitutively expressing Bcl xL and suggests that variation of its expression in response to treatment method too as variation of your activation of its professional apoptotic partners may very well be very important determinants of chemosensitivity. As a way to build new targeted approaches, this kind of qualities may possibly nicely be even more very important to check out compared to the prognostic value from the basal expression degree with the protein.

The most crucial event within the regulation of angiogenesis stands out as the signaling cascade that requires vascular endothelial growth element . VEGF A could be the predominant proangiogenic molecule that stimulates endothelial cells to secrete cytokines necessary for cellmigration and sprouting of newvessels for your induction of the two angiogenesis and lymphangiogenesis in tumor cells . Many studies have evaluated the prognostic significance of invasion and angiogenesis relevant genes in breast cancer. Having said that, it is vital to correlate expression amounts with all the histological grade in premenopausal breast tumors that are inclined to be frequently alot more aggressive resulting from greater tumor size, higher numbers of metastatic lymph nodes, lower rates of hormone receptor good standing, earlier and more regular locoregional recurrences and poorer general survival than breast tumors that create in gals soon after menopause . Previously we reported aberrant NF ?B signaling and apoptosis evasion that was additional important in premenopausal relative to postmenopausal sufferers .
The existing study was thus designed to investigate the expression of MMP TIMP , RECK, HIF and VEGF in premenopausal breast cancer patients of different histological grades. Components selleck chemical purchase PS-341 and approaches Chemical compounds Acrylamide, bovine serum albumin , bromophenol blue, mercaptoethanol, N,N,N ,N tetramethylene diamine , N,N methylene bisacrylamide, proteinase K, and sodium dodecylsulphate , were purchased from Sigma Chemical Organization . All other reagents utilised had been of analytical grade. Review population Seventy five newly diagnosed breast cancer individuals, mean age years from Welcare Hospital, Vytilla, Kerala, India, who had not undergone any prior treatment for his or her tumors have been picked for that review. They had been histologically categorized as grades I, II, and III and in accordance to hormonal standing as premenopausal patients. The individuals had been not implementing hormones or oral contraceptives and have been nonsmokers.
None of them had concomitant disorders which include diabetes mellitus, liver illnesses, and rheumatoid arthritis. The individuals had been eligible if they have been menstruating on a regular basis, not pregnant or breastfeeding, and had not undergone hysterectomy or oophorectomy. Informed consent was obtained from all the participants. The Human Ethics Committee, India accepted the review. The histological JNJ 26854165 grading was based on Scarff Bloom Richardson’s classification as modified by Elston and Ellis . Fresh tumor tissues along with a rim of cm of healthier adjacent breast tissue throughout the malignant breast lesion have been subdivided and variously processed for distribution to each and every experiment.

Immunohistochemical analysis in the eggs showed disorganized microtubules and chromosomal misalignment followed by a failure of PB emission . Within the current research, we demonstrated that Thr and Ser phosphorylated Akts perform individually. Though the inhibition of both of your Akts resulted within a shorter spindle, the distribution of Thr and Ser phosphorylated Akts is thoroughly various in MII oocytes. To determine whether Thr and Ser phosphorylated Akts have distinctive functions during the fertilization of MII oocytes, we performed in vitro fertilization utilizing oocytes taken care of with peptide at MII for h. While oocytes exhibited PB emission after the injection of the peptide for Thr phosphorylated Akts, the chromosomal alignment and microtubular organization have been aberrant . In contrast, the injection of a peptide for Ser phosphorylated Akt brought about a failure of PB emission . These final results recommend that person Thr and Ser phosphorylated Akt actions are involved in fertilization to complete meiosis.
Our present final results also propose that two active types have unique roles, i.e Ser phosphorylated pop over to this website Akt action is involved in PB emission although Thr regulates the organization of microtubules. Substantial level Akt expression while in meiotic maturation disappeared while in pre implantation advancement Making use of immunohistochemical examination, we’ve got previously demonstrated that Akt is expressed in the course of meiotic maturation . While in the existing research, our final results suggest that Akt disappears following fertilization. To tackle whether the Akt protein is re expressed, we examined the expression of Akt protein and mRNA during oocyte meiotic maturation and embryonic advancement. By Western blot analysis, similarly large levels of phosphorylated and total Akts have been detected from GV to MII for the duration of meiotic maturation . Akt has three isoforms which are differentially expressed within a selection of tissues . As illustrated in Fig. B, Akt and Akt mRNA were expressed whereas Akt mRNA was not detected.
These effects propose that Akt and Akt are involved in spindle perform and PB emission throughout meiotic maturation. In contrast for the oocytes, complete Akt protein and mRNA in embryos have been expressed at particularly low to undetectable ranges whatsoever phases of pre implantation development . These effects propose that Akt function inside the spindle is oocyte particular, to finish meiotic maturation by PB emission. Discussion We now have previously demonstrated 850649-62-6 SYR-322 that publicity to LY, an inhibitor of PIK exercise, resulted in quite reduced to undetectable ranges of Thr phosphorylated Akt and an aberrant distribution of Ser phosphorylated Akt at MI in oocytes . In the current research, our benefits uncovered the inhibition of Akt induced incomplete GVBD followed by a failure of MI. These benefits suggest that comprehensive GVBD is dependent on Akt acting via PIK.

PGE may perhaps induce VEGF expression by way of binding for the EP receptor and activating the JNK and HIF pathstrategies . PGE has also been proven to boost binding of endothelial cells for the extracellular matrix by way of V dependent mechanisms . In summary, the selective COX inhibitor DuP has become located to induce apoptosis and avoid capillary like tubule formation in vitro at pharmacologically appropriate concentrations. The results observed may well perhaps be attributable to the distinct inhibition of COX by DuP with a subsequent decrease in PGE manufacturing. In addition, our information has demonstrated that DuP induced apoptosis in HUVECs may perhaps be caspasedependent when the inhibition of tubule formation could possibly come about by way of a caspase independent mechanism. The transforming development aspect family members includes three closely associated isoforms which have been prototypes from the more substantial TGF superfamily . TGF family members elicit a various choice of cellular responses which include cell proliferation, migration, fibrosis, irritation, andwound restore .
The biological functions of TGF are of widespread relevance in embryogenesis and in fibroproliferative ailments in adults, such as, liver cirrhosis, chronic glomerulonephritis, and atherosclerosis . TGF knockout mice develop diffuse mononuclear cell infiltrates that prove lethal inside a handful of weeks of birth . Latest studies have suggested a fundamental position for TGF as being a significant mediator in the tissue response to Seliciclib Roscovitine lung damage . In addition, TGF plays a protective part in attenuating cellular harm and sustaining tissue homeostasis as a result of induction of cytoprotective proteins this kind of as heme oxygenase . HO plays significant roles in physiological iron homeostasis, antioxidant defense, anti irritation, and antiapoptotic results . HO catalyzes the conversion of heme to biliverdin, releasing equimolar quantities of carbon monoxide and iron. Subsequently, biliverdin is converted to bilirubin by biliverdin reductase . 3 isoforms of HO are identified . HO is surely an inducible enzyme which is most really concentrated in tissues which have been heavily involved with the catabolism of heme proteins .
HO is known as a non inducible isoform that’s existing in highest concentrations inside the brain and testes and is thought Bergenin to get particularly involved with signaling pathways .HO is surely an isoformwith low catalytic activity and its physiological purpose is uncertain . HO is induced by heme solutions and also a wide wide range of non heme stimuli, which incorporate oxidant pressure and TGF . A variety of consensus sequences, like people for nuclear issue ?B , activating protein , and heat shock responsive component within the area within the HO gene, are identified as regulatory sequences that can induce HO expression in response to several stimuli . NF ?B, a significant transcription element for regulating HO expression, is often a dimer on the transcription components p and p .

The proteins were then visualized working with an enhanced chemiluminescence detection kit siRNA transfection To knockdownATM expression, synthetic ATMsiRNA duplex oligomer as well as a scrambled siRNA duplex oligomer were bought from Utilized Biosystems. For siRNA transfection experiments, A cells were plated onto mmdishes and cultured overnight in total medium. The following morning, cells were transiently transfected with Oligofectamine supplemented with ATM siRNA . At h publish transfection, cells have been treated with or devoid of emodin for an alternative h. Cells had been then harvested for detection the protein degree of ATM by immunoblotting Total RNA planning and RT PCR evaluation The cells were trypsinized and washed twice with PBS. Total RNA was ready using a Qiagen RNA extraction kit. The RNA concentration was established by studying the absorbance at and nm that has a UV spectrophotometer. A complete of g of cDNA was synthesized as outlined by the manufacturer’s instructions . For PCR amplification of distinct genes, a reaction mixture that contained M dNTP, mM MgCl, pmol primers, unit of Taq polymerase and g of cDNA product or service was ready on ice.
PCR was performed in the exponential range, plus the PCR solutions had been separated by electrophoresis on agarose gels stained with ethidium bromide and analyzed applying the Ever Gene Image Method. actin gene was analyzed as an internal loading handle. The measurement of mitochondrial membrane potentials and reactive oxygen species generation had been carried out as previously described . Briefly, A cells had been handled Odanacatib with or without having M emodin at the indicated time points. Just after therapy, the cells were incubated with , dichlorodihydrofluorescein diacetate , dihydroethidine or JC at C for one more min. The cells had been then washed 3 times using a cold PBS choice, as well as the fluorescence intensity of the cells was analyzed utilizing a Becton Dickinson Movement cytometer. Statistical evaluation The many figures proven within this articlewere obtained fromat least 3 independent experimentswith very similar benefits. All information are presented as imply S.E.M. of at least 3 separate experiments. Statistical differences were evaluated working with the Student’s t test and viewed as considerable at P P .
or P . Final results Emodin treatment method induces selleck chemicals article source the accumulation of p and Bax, but decreases the expression of survivin We previously demonstrated that emodin could selectively destroy human lung adenocarcinoma A cells, but not non tumor cells for instance human fibroblast like lung WI cells, by activating a reactive oxygen species dependent mitochondrial pathway . In addition, emodin exerts anti tumorigenic exercise by inducing apoptosis in diverse cancer cells. Because emodin is demonstrated to be a genotoxic compound, and simply because most cytotoxic drugs induce apoptosis by activating the p dependent pathway, we investigated no matter whether p plays a part in emodin triggered apoptosis in human lung adenocarcinoma A cells.

Phosphorylation at these online sites results while in the binding of Awful to proteins and inhibition of the binding of Bad to Bcl and Bcl xL . Akt has become shown to advertise cell survival through its ability to phosphorylate Bad at Ser . Our success demonstrated that the expression levels within the proapoptotic proteins Bax and Poor did not change significantly, whereas the expression amounts on the anti apoptotic proteins Bcl and Bcl xL declined in a time dependent method, which signifies that the regulation of expression with the Bcl protein family may perhaps contribute to BJ B induced apoptosis . Importantly, the expression degree of p Negative at Ser was suppressed, which suggests that BJ B induced mitochondrial dysfunction may be related to the Akt signaling pathway. The Akt pathways are very well characterized cell survival signaling pathways that block apoptosis inside a variety of cell sorts . Akt has a broad variety of downstream targets that regulate tumor connected cell processes this kind of as cell development, cell cycle progression, survival, migration, epithelial mesenchymal transition, and angiogenesis.
Blockade of Akt signaling results in apoptosis and development inhibition of tumor cells . CML is often a malignant disorder that success through the neoplastic transformation of a hematopoietic stem cell , which carries the t translocation accountable for expression of the fusion gene Bcr Abl, a constitutively energetic tyrosine kinase . Bcr Abl exerts selleck chemical discover this its oncogenic perform by activation of the cascade of intracellular signaling pathways, which include things like MAPK, PIK Akt and Jak STAT, that ultimately result in elevated survival and proliferation, and avoidance of apoptosis . Bcr Abl and Akt are both listed inside the Hsp client proteins which could be degraded by a Hsp inhibitor . In this examine, we have now noticed that BJ B lowered expression of Bcr Abl to nearly undetectable levels. Additionally, it lowered expression from the Akt protein and induced vital Akt dephosphorylation . We speculate that BJ B led to degradation of Bcr Abl and decomposition of p Bcr Abl complexes, which even further downregulated the expression ranges of Akt and p Akt.
There is also a possibility that Akt and p Akt are immediately degraded by BJ B inhibition of Hsp. Additionally, co IP and Western blot analyses were applied to find out the interaction between Bcl xL and Negative, whose phosphorylation is controlled through the Akt pathway . BJ B induced the dissociation of Negative from and even more Bcl xL was observed to interact with Lousy , which supplied even further Dapagliflozin support to our hypothesis that BJ B induced mitochondrial dysfunction might be related to inactivation with the Akt signaling pathway. Based on the results of this study along with the readily available literature, many different pathways by which BJ B may possibly consequence in apoptotic cell death are advised, as proven during the composite scheme in Fig Within the current review, we mostly analyzed the molecular mechanism of BJ B induced apoptosis in human CML K cells.

Two way ANOVA was put to use to confirm that no interaction existed between different remedies and distinctive experiments. Benefits TNF a and butyrate act synergistically to induce apoptosis of CaCo colonic epithelial cells Caco cells were refractory towards the induction of apoptosis by recombinant human TNF a, as much as ng ml ; nonetheless, co administration of mM sodium butyrate resulted in considerable apoptosis, assessed about the basis of nuclear morphology. Fig. exhibits the percentage of apoptotic cells, h just after treatment with TNF a , in the presence or absence of mM butyrate. Apoptosis was significantly greater in TNF a butyrate handled cultures , than in individuals taken care of with either agent alone or in handle cultures . Reduction in viable cell amount measured over h , was greater in TNF a butyrate treated cells than in cultures taken care of with either agent alone . Butyrate alone also resulted within a major reduce in viable cell variety just after h , but this was nonetheless appreciably less than the reduction noticed following TNF a butyrate co treatment.
TNF a butyrate induced apoptosis is connected to DNA fragmentation, processing of caspase and increased numbers of cells with sub G http://www.selleckchem.com/products/Vatalanib,Dihydrochloride-Salt.html phase DNA articles Apoptosis in response to TNF a butyrate was confirmed by labelling of DNA strand breaks, applying the TUNEL based, TdT in situ assay kit modified for fluorescence microscopy . Also, Western blotting and immunofluorescence had been applied to confirm the processing of caspase in TNF a butyratetreated cells . Flow cytometric analysis demonstrated that remedy of CaCo cells with both TNF a butyrate, or butyrate alone, resulted inside a vital decrease in the proportion of cells inside the G phase of the cell cycle . At this time, there was also a significant improve inside the proportion of cells with lower than n DNA material , in TNF a butyrate handled cultures, but not in people treated with butyrate alone . By h, each butyrate alone and TNF a butyrate resulted in considerable increases in sub G cells ; on the other hand, apoptotis was significantly better in response to TNF a butyrate than to butyrate alone.
MGCD-265 Inhibition of each caspase and caspase ameliorates TNF a butyrate apoptosis Caspase is established because the proximal caspase within the apoptotic signalling cascade activated in response to death receptor ligation. Lately, caspase is recognized as taking part in a comparable role . The expression of both caspase and caspase in CaCo cells was demonstrated by Western blotting . Consequently, the action of specific inhibitors for both caspase and caspase have been tested, alone and in combination. Experiments had been carried out to examine the effect of pre treatment with z IETD.fmk and z AEVD.fmk , alone or in combination, on apoptosis measured by TUNEL and nuclear morphology, h after treatment of cells with TNF a butyrate .

Additionally, determination of your total COX ranges by FACS examination in various melanoma cell lines confirmed presence of high ranges of COX in WM and LOX cells and common ranges in LU and WM cells . Certain inhibition of COX activity by NS alone had no substantial effects on induction of apoptosis in melanoma cells. Then again, mixed treatment method with sodium arsenite and NS synergistically greater apoptosis in Fas favourable melanomas WM, LU, WM and LOX h and h after therapy . Total amounts of cell death of melanomas induced by mixed treatment of sodium arsenite and NS had been somewhat larger than apoptotic amounts resulting from the secondary necrosis . To assess a probable role from the FasL Fas mediated death in arsenite and NS treated melanomas, we to start with established ranges of surface expression of Fas and FasL following this kind of remedy. We observed a marginal impact around the surface Fas receptor ranges just after therapy of melanomas with arsenite and NS. TNF stimulation was utilized like a constructive handle for upregulation of Fas ranges .
In contrast, the surface amounts of FasL had been notably increased h immediately after mixed therapy with sodium arsenite and NS in WM, LU , WM and LOX melanoma cells . Arsenite or NS alone didn’t induce a notable expression of FasL to the cell surface . Anti FasL inhibitory mAb partially suppressed apoptosis induced with arsenite and NS in all melanoma lines examined, whereas result of anti TNF mAb was selleck chemicals more hints pronounced only in WM cells . This impact of anti TNF mAb on WM cells was very likely on account of inhibition of arsenite induced TNF mediated apoptosis in these cells . To demonstrate a dependence of apoptosis induced by arsenite and NS on caspase routines, we used certain inhibitors of caspases. Each Ac IETD CHO and Ac LEHD CHO partially suppressed arsenite and NS induced apoptosis, even though Ac IETD CHO was additional successful , indicating that death receptor caspase mediated cascade operated in the course of apoptosis. A standard caspase inhibitor, zVAD fmk , was very effective for suppression of apoptosis, though this suppression was not finish, probable because of secondary necrosis .
Pazopanib Taken with each other, these data demonstrated that the upregulation from the surface FasL expression in many melanoma lines following the combined remedy with arsenite and COX inhibitor could potentially explain an increase while in the apoptotic response. Therefore, in addition to basal apoptosis driven by sodium arsenite , combined remedy with sodium arsenite and NS induced FasL Fas mediated apoptosis in melanoma cells. Regulation of the FasL expression by combined therapy with sodium arsenite and NS There are quite a few conceivable targets for modulation of FasL expression for the cell surface: the FasL promoter exercise and subsequent transcription and translation; posttranslational modifications of FasL; FasL protein translocation through the cytoplasmic pool by secretory lysosomes for the cell surface; membrane FasL internalization and degradation; membrane FasL cleavage to the cell surface by matrix metalloproteinases .

Morphology, growth, apoptotic, and senescence scientific studies on MCF As MCF As cells have uniform and basal epithelial morphology, dimension, and form at regular and identical growth problems. Data also imply typical anchorage dependent development of these cells in tissue culture dishes. In spite of p being a regulator of senescence and differentiation and MCF As cells having negligible total p, these tend not to express cellular senescence connected galactosidase and consequently are certainly not senescent even right after currently being in culture for weeks . The doxorubicin treated MCF cells are shown as beneficial management to the approach employed . We additional investigated the growth pattern by performing MTT proliferation assay as described in Supplies and methods. As proven in Fig. B, MCF As cells grow additional rapidly than parental MCF cells. The doubling time of MCF As was about h when compared to N h for MCF . MCF As cells have proliferative phenotype because of upregulated cyclin D and overexpression of p downregulates cyclin D MCF As cells were identical to MCF cells except for your growth pattern as indicated by MTT proliferation assay .
As proven in Fig. C, the altered selleckchem tsa inhibitor growth charge of MCF As is due to variations in distribution of cells in different phases of cell cycle. The cell cycle analysis by flowcytometry uncovered that in MCF As cells G G was considerably depleted and more cells accumulated in S GM phases inside h of standard growth circumstances. Also, no change in sub G G population that designates apoptotic phenotype was detected in MCF As cells. Additionally, to investigate regardless of whether there exists any alteration during the status of cyclins that management cell cycle phase transitions and also regulate its progression, we investigated the standing of cyclin D and cyclin E. Both MCF As and MCF cells had been serum starved for h. As shown in Fig. A, cyclin D was barely detectable in MCF cells whereas in MCF As cells considerably increased expression of cyclin D was detected. Following h serum starvation, the cells had been more grown in media supplemented with serum for and h.
As could very well be noticed, cyclin D was detected in MCF at the same time as MCF As cells . Then again, at any provided time point cyclin D levels in hop over to this site MCF As cells are considerably increased than people in MCF cells. Increase in cyclin D expression in MCF As cells was further reconfirmed by confocal microscopy studies . Beneath very similar experimental circumstances no major alterations in either cyclin E or actin have been detected in the two the cell lines. In MCF As cells given that cyclin D is overexpressed, it can be very likely that this big difference may be attributed to enhanced growth of these cells.

In this way we detected endogenous SMC and tubulin interacting with the His PH in . Detection of SMC, Zizimin, PLC? and tubulin within a complicated with PH domain of Bcr Abl protein confirms our proteomics information and suggests that the Bcr Abl PH domain may be associated with multifunctional intracellular pursuits, which include regulation of cytoskeleton, cell metabolic process and signaling transduction. Lipid binding profile in the Bc Abl PH domain According on the latest paradigm, PH domains mostly perform as protein anchors to your plasma membrane . To investigate the lipid binding specificity, purified His tagged Bcr PH domain was incubated together with nitrocellulose filter pre spotted with several phospholipids and an anti His antibody was utilised to probe the membranes for protein binding . Protein tag encoded by empty vector was made use of like a adverse handle to define feasible non certain binding . Within this assay, PH domain specifically bound to PtdIns P, PtdIns P, PtdIns P. For your subsequent experiment, we applied PIP Array membrane prespotted by using a concentration gradient of lipids.
This assay confirmed the PH domain binds to all 3 of the monophosphates with higher affinity . The ability to realize monophosphates exceptionally is very uncommon in PH relatives. It’s been recommended that only of PH loved ones have substantial specificity of binding lipids, largely di and thrisphosphates selleckchem ATP-competitive Raf inhibitor . It is actually nicely established that there is uneven distribution of phosphainositides from the cell. So, binding to particular lipids determines the localization of PH containing protein. As an example, PtdIns P is an abundant part in the Golgi membrane , whereas PtdIns P can be a part of early endosome membrane and plays important position in endocytosis . To find out the main difference of cell localization of p and p Bcr Abl proteins, Cos cells had been transfected by corresponding constructs expressing the two proteins. Cells have been stained by anti Abl antibodies followed by anti GM antibodies to visualize the Golgi complicated.
p Bcr Abl was localized in the perinuclear place and overlapped with the GM staining Acetylcysteine suggesting that it possessed the capability to bind to your Golgi membrane as a result of its PH domain . In contrast, p Bcr Abl localized alot more uniformly within the cytoplasm. We following handled the cells with M Wortmannin h prior to fixation. This compound is really a renowned inhibitor of PIK but, at increased concentrations, also of PIK . Interestingly, Wortmannin treatment interfered using the Golgi localization of p Bcr Abl, which was discovered to get localized on the cytoplasm just like p Bcr Abl . Also, we taken care of cells with plasmids encoding shRNAs precise for PIK and PTEN. For these experiments we made use of human HEK cells and we to begin with confirmed the efficiency of the shRNAs by analyzing cells transfected with shRNAs by immunoblotting or true time PCR .