scholarly journals Hypoxia and its possible relationship with endometrial receptivity in adenomyosis: a preliminary study

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Song Guo ◽  
Di Zhang ◽  
Xiaowei Lu ◽  
Qian Zhang ◽  
Ruihuan Gu ◽  
...  
Keyword(s):  
Real Time ◽  
Western Blotting ◽  
Real Time Pcr ◽  
Underlying Mechanism ◽  
Endometrial Receptivity ◽  
Control Group ◽  
Expression Levels ◽  
Preliminary Study

Abstract Background Adenomyosis (AM) is an important cause of female infertility. However, the underlying mechanism remains unclear. This report describes a preliminary study of hypoxia and its possible association with endometrial receptivity in AM. Methods The study was divided into in vitro and in vivo experiments. In vitro, expression levels of the endometrial receptivity markers HOXA10 and HOXA11 in the implantation period were examined using real-time PCR and western blotting. Endometrial expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, and HIF-3α was determined using immunohistochemistry. In vivo, using an AM mouse model established by oral administration of tamoxifen, we inhibited expression of HIF-2α using an HIF-2α antagonist (PT2399; 30 mg/kg body weight, twice daily by oral gavage for 2 days) and then examined expression levels of Hoxa10 and Hoxa11 using real-time PCR and western blotting. Results Endometrial mRNA and protein expression levels of HOXA10 and HOXA11 were significantly lower in patients with AM than in control patients. Expression of HIF-2α was significantly higher in the AM group than in the control group, whereas that of HIF-1α and HIF-3α was equivalent in both groups. In vivo analysis showed that administration of the HIF-2α antagonist resulted in increased expression of Hoxa10 and Hoxa11 at both the mRNA and protein levels in AM model mice. Conclusions HIF-2α overexpression may be one reason for decreased endometrial receptivity in AM. The current findings provide insight into HIF-2α-mediated AM-related infertility and suggest that PT2399 has potential as a treatment for AM. Trial registration This trial was retrospectively registered.

232 EFFECT OF SUPEROVULATION PRETREATMENT ON DEVELOPMENTAL CHARACTERISTICS OF IN VITRO-FERTILIZED BOVINE EMBRYOS TRANSFERRED TO THE OVIDUCT-UTERUS ENVIRONMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 247
Author(s):  
V. Havlicek ◽  
A. Gad ◽  
S. Papp ◽  
K. Stein ◽  
F. Palm ◽  
...  
Keyword(s):  
Real Time ◽  
Embryo Development ◽  
Internal Control ◽  
Real Time Pcr ◽  
Control Group ◽  
Hormonal Changes ◽  
Expression Levels ◽  
In Vivo Culture

Superovulation is a routine procedure to stimulate growth and ovulation of multiple follicles. However, the hormonal changes in the reproductive tract after superovulation treatment affect the environment and subsequently the early embryo development. The aim of the study was to examine the effect of superovulation pretreatment on embryo development and gene expression of IVM/IVF derived embryos subsequently cultured in vivo. The cumulus‐oocyte complexes derived from slaughterhouse ovaries were in vitro matured and fertilized. The denuded presumptive zygotes were cultured in CR1 medium with 5% oestrous cow serum. A total of 788 cleaved embryos at Day 2 were transferred by transvaginal endoscopy into the oviduct of synchronized and superovulated heifers (superstimulated group, SS) and 784 cleaved embryos were transferred into the ipsilateral oviduct of single ovulated synchronized heifers (single ovulation group, SO). In total, 10 Simmental heifers were used for in vivo culture in a crossover design. The in vivo culture was repeated once at an interval of at least 6 weeks in the same animal. At Day 7, embryos were recovered by combined flushing of the oviducts by endoscopy and the adjacent part of the uterine horns by conventional procedure. The numbers of recovered blastocysts were recorded and the embryos were cultured for the following 48 h to determine the blastocyst rate at Days 8 and 9. Simultaneously, 410 cleaved embryos were cultured in vitro for 9 days (control group, C). Triplicate pools of 10 blastocysts recovered at Day 7 from each treatment group were used for RNA isolation. Real-time PCR using sequence specific primers was performed in StepOnePlus™ real time PCR system (Applied Biosystem, Foster City, CA, USA). A comparative threshold cycle method was used to quantify expression levels of the candidate genes compared to the internal control GAPDH gene. The number of recovered embryos after in vivo culture was significantly lower in the SS group compared with the SO group (66.9 v. 79.5%, respectively; P < 0.05). The blastocyst rates at Days 7, 8, and 9 in the SS, SO, and C groups were not significantly different (31.9, 43.3, and 47.1% v. 35.2, 48.5, and 53.5% v. 37.8, 50, and 56.1%, respectively). Molecular analysis of selected genes playing important roles during pre-implantation development revealed significantly lower expression levels of IL6, IL18, and ABCC2 between both experimental in vivo culture groups and the C-group. The IL18 was also significantly down-regulated in the SS-group compared to the SO-group. The transcription factor NFκB was found to be down-regulated in the SS-group compared to the SO and C groups (P < 0.05). In conclusion, we showed that the superovulation pretreatment did not affect blastocyst yield during the culture period but seemed to influence the expression of developmentally important genes in the resulting embryos.

scholarly journals The Effect of Low Intensity Pulsed Ultrasound on Endometrial Receptivity in Infertile Patients with Adenomyosis

2022 ◽  
Author(s):  
Ling Long ◽  
Ling Zhou ◽  
Jing Zhu ◽  
Xuan He
Keyword(s):  
Western Blotting ◽  
Endometrial Receptivity ◽  
Control Group ◽  
Pulsed Ultrasound ◽  
Expression Levels ◽  
Low Intensity Pulsed Ultrasound ◽  
Low Intensity ◽  
Infertile Patients

Abstract Objective: Adenomyosis (AM) is an important cause of female infertility, and its disease mechanism remains unclear. This study preliminarily investigated the expression of endometrial receptivity markers homeobox A10 (HOXA10) and leukemia inhibitory factor (LIF) in infertile patients with AM and described the effects of low intensity pulsed ultrasound (LIPUS) on it. Methods: In vivo, tissues were obtained from the infertile female AM patient group (AG group, n=10) and healthy control group (CG group, n=11). The expression of HOXA10 and LIF in the two groups was detected by immunohistochemistry (IHC) and western blotting. In vitro, primary cells were extracted and cultured from the two groups, and the expression of HOXA10 and LIF protein was detected by western blotting. Then the AG cells were treated with 15, 30, and 60 mW/cm2 of LIPUS for 7 days (20 min/day), and detected the cell adhesion rate. Finally, treat the AG cells with 30mW/cm2 LIPUS for 7 days (20 min/day), and detect the expression level of ICAM-1 in the cell supernatant by ELISA. The AG cells was treated with 30 mW/cm2 LIPUS for 4 days (20 min/day), and the expression levels of HOXA10 and LIF were detected by western blotting, RT-PCR, and agarose gel electrophoresis. Results: In vivo, IHC staining showed that HOXA10 and LIF proteins were mainly localized in endometrial epithelial cells. Both IHC and western blot showed that the levels of HOXA10 and LIF in the AG group were significantly lower than those in the CG group (P<0.01, P<0.05). In vitro, the expression levels of HOXA10 and LIF protein in the AG cell was significantly lower than those in the CG cell (P<0.001). Then, the cell adhesion ability of the 30 and 60 mW/cm2 groups was higher than that of the 15 mW/cm2 group after LIPUS treatment. Finally, The concentration of ICAM-1 in the supernatant of AG cells treated with LIPUS was significantly higher than that of the control group (P<0.01), and the AG cells were treated with 30 mW/cm2 LIPUS for 4 days (20 min/day), the protein and mRNA expression levels of HOXA10 and LIF were higher than those of the control group (P<0.001). Conclusion: The reduction of HOXA10 and LIF may be one of the reasons for the decreased endometrial receptivity in AM. The LIPUS promoted the adhesion and the expression of HOXA10 and LIF of EEECs from the AM group, thereby increasing endometrial receptivity.

scholarly journals Fuyuan Xingnao Decoction Promotes Angiogenesis Through the Rab1/AT1R Pathway in Diabetes Mellitus Complicated With Cerebral Infarction

2021 ◽  
Vol 12 ◽  
Author(s):  
Dong Deng ◽  
Yao Qu ◽  
Lihua Sun ◽  
Liyang Jia ◽  
Jianhong Bu ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Cerebral Cortex ◽  
Cerebral Infarction ◽  
Real Time ◽  
Control Group ◽  
Ang Ii ◽  
Cerebral Microvessels ◽  
Expression Levels

Fuyuan Xingnao decoction (FYXN), a traditional Chinese formula comprised of seven herbs, has been utilized to treat diabetes mellitus complicated with cerebral infarction (DMCI) for years. Yet, its protective and regulatory mechanism is poorly understood. The aim of the study is to investigate the effects of FYXN on DMCI in vitro and in vivo, as well as its mechanism in angiogenesis. For in vivo experiments, FYXN was administered to DMCI rats with streptozotocin (STZ) injection-induced diabetes. Then middle cerebral artery occlusion (MCAO) was conducted and the cerebral cortex sections of the rats were obtained. The ultrastructure of cerebral microvessels and new vessel density of ischemic penumbra were evaluated by the transmission electron microscopy (TEM) assay and immunohistochemistry, respectively. Protein and mRNA expression levels of Rab1/AT1R in cortex were assayed by Western blotting and real-time fluorescence quantitative real-time polymerase chain reaction (RT-qPCR). In vitro, FYXN serum was produced in rats on the fourth day 2 h after the last FYXN administration. Green fluorescence was observed after transfection with lentivirus packaged Rab1-WT or siRNA for 24 h. The activity of brain microvascular endothelial cells (BMECs) treated with sera from these rats was tested by MTT assay and Transwell assays, respectively. The expression of AT1R on the cell membrane and endoplasmic reticulum of BMECs was evaluated by immunofluorescence staining. Protein expression levels of signaling molecules in the Rab1/AT1R pathways were also detected. Results showed that in vivo, FYXN treatment significantly intensified CD31 staining in the cortical areas and enhanced the mRNA and protein levels of AT1R, Ang II, Rab1a, Rab1b and VEGF expression in ischemic cerebral cortex tissues. In vitro, the expression levels of AT1R, Ang II, Rab1a, Rab1b and VEGF in the cerebral infarction model group were significantly higher than those in the control group, with further increases after administration of FYXN drug serum. FYXN promoted the proliferation and migration of BMECs by activating the Rab1/AT1R signaling pathway. In conclusion, FYXN exerts a protective effect against DMCI by promoting angiogenesis via the Rab1/AT1R pathway, which provides strong evidence for the therapeutic effect of FYXN on DMCI.

scholarly journals The Effects of miR-136-5p-Mediated Regulation of A20 in Astrocytes from Cultured Spinal Cord Cultured Cells In Vitro

2017 ◽  
Vol 41 (4) ◽  
pp. 1596-1604 ◽  
Author(s):  
Xiaoming Peng ◽  
Xiongzhi Shi ◽  
Jinmin Zhao ◽  
Jichen He ◽  
Keke Li ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Inflammatory Cytokine ◽  
Cultured Cells ◽  
Expression Level ◽  
Control Group ◽  
Interleukin 17 ◽  
Expression Levels ◽  
Inhibition Group

Background/Aims: This study focused on investigating the regulatory mechanism of miR-136-5p in mouse astrocytes stimulated with interleukin-17(IL-17). Methods: C57BL/6 mouse astrocytes were stimulated with IL-17 (100ng/ml) for various periods of time (0-48 hours) and at various doses (0-200 ng), and the expression levels of inflammatory cytokine and chemokine genes (IL-6, TNF-α, MCP-1, MCP-5 and MIP-2) were then detected by real-time PCR. The expression of the A20 gene was measured with real-time PCR in cells that were stimulated with IL-17 (50 ng/ml) for various periods of time (0-48 hours). C57BL/6 mouse astrocytes were transfected with Ctrl-anti-miR-136-5p or LNA -anti-miR-136-5p for 48 h. Thereafter, the cells were stimulated with or without IL-17 (50ng/ml) for 6 h. The level of A20 protein (TNFα-induced protein 3, TNFAIP3) was detected by Western blot analysis. Results: (1) Compared with the DMEM control group, within six hours, IL-17 stimulation significantly increased the expression levels of inflammatory cytokine and chemokine genes and clearly decreased the expression level of the A20 protein. (2) Without IL-17 stimulation, the expression level of the miR-136-5p gene was significantly decreased, whereas in the miR-136-5p-inhibition group, the A20 protein expression was elevated. IL-17 stimulation slightly decreased the expression of the A20 protein in the miR-136-5p-inhibition group, but it was still slightly higher than in the control group. Conclusion: This study demonstrated that miR-136-5p affected the expression of A20 in IL-17-stimulated astrocytes.

253 DIFFERENTIAL GENE EXPRESSION IN IN VIVO-DERIVED v. IN VITRO-PRODUCED EQUINE BLASTOCYSTS AS DETERMINED BY RT-qPCR

2010 ◽  
Vol 22 (1) ◽  
pp. 284
Author(s):  
K. Smits ◽  
K. Goossens ◽  
A. Van Soom ◽  
L. Peelman
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Differential Gene Expression ◽  
Real Time Pcr ◽  
Calf Serum ◽  
Fatty Acid Binding ◽  
Expression Levels ◽  
Differential Gene

Although in vitro production of equine embryos has greatly evolved in recent years, there are still substantial differences between in vitro-produced and in vivo-derived equine embryos. Fundamental insight into these differences could lead to optimization of equine assisted reproductive techniques. Reverse transcription quantitative real-time PCR (RT-qPCR) is a highly specific and sensitive tool to compare mRNA expression levels of specific genes and was used in this study to determine differences in gene expression between equine in vivo and in vitro embryos. In vivo embryos (n = 8) were derived by uterine flushing of artificially inseminated mares at 7 days after ovulation. For the production of the in vitro embryos (n = 8), oocytes from slaughtered mares were matured in DMEM-F12-based medium (Galli et al. 2007 Anim. Reprod. Sci. 98, 39-55) in 5% CO2 in air (maturation rate: 57%), fertilized by intracytoplasmic sperm injection, and cultured in DMEM-F12 with 10% fetal calf serum in 5% CO2, 5% O2, and 90% N2 for 9.5 days (cleavage rate: 74%; blastocyst rate: 7%). RNA was extracted from single early to expanded blastocysts and amplified and converted into cDNA with the WT-Ovation RNA Amplification System (NuGEN, San Carlos, CA, USA). Based on the presumed gene functions and differential gene expression as determined in a previously performed suppression subtractive hybridization (SSH; Smits et al. 2009 Reprod. Dom. Anim. 44, 75), 5 genes [brain expressed X-linked 2 (BEX2), Mps one binder kinase activator-like 3 (MOBKL3), fatty acid binding protein 3 (FABP3), minichromosome maintenance complex component 7 (MCM7), and ornithine decarboxylase (ODC)] were selected for quantification by RT-qPCR with the KAPA SYBR® FAST qPCR Kit (Kapa Biosystems, Belgium) on the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Nazareth, Belgium). All data were normalized with previously determined stable reference genes (beta actin, ubiquitin C, ribosomal protein L32, and glyceraldehyde-3-phosphate dehydrogenase) and statistically analyzed by means of a Mann-Whitney test. The fact that all genes were expressed at greater levels in the in vivo-derived blastocysts than in the in vitro-produced blastocysts confirmed the results of the SSH. This difference was highly significant for MOBKL3, BEX2, and ODC (P < 0.005), significant for FABP3 (P < 0.05), and not significant for MCM7. These genes have already been shown to be important for embryonic cell survival (ODC), oocyte maturation and pre- implantation development (MOBKL3) in mice, regulation during embryonic development (BEX2) and fetal development (FABP3) in human, and genome replication in eukaryotes (MCM7) (Pendeville et al. 2001 Mol. Cell Biol. 21, 6549-6558; Han et al. 2005 Nucleic Acids Res. 33, 6555-6565). In conclusion, 4 genes (MOBKL3, BEX2, ODC, and FABP3) with greater expression levels in in vivo-derived equine blastocysts have been identified. Whether the up-regulation of these genes is important for normal embryonic differentiation in the horse embryo is currently under investigation.

scholarly journals Effects of Naringin on Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells In Vitro and In Vivo

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Lihua Yin ◽  
Wenxiao Cheng ◽  
Zishun Qin ◽  
Hongdou Yu ◽  
Zhanhai Yu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Trabecular Structure ◽  
Control Group ◽  
Periodontal Ligament Stem Cells ◽  
Expression Levels ◽  
Proliferation And Differentiation

This study is to explore the osteogenesis potential of the human periodontal ligament stem cells (hPDLSCs) induced by naringin in vitro and in vitro. The results confirmed that 1 μM naringin performs the best effect and a collection of bone-related genes (RUNX2,COL1A2, OPN, and OCN) had significantly higher expression levels compared to the control group. Furthermore, a typical trabecular structure was observed in vivo, surrounded by a large amount of osteoblasts. These results demonstrated that naringin, at a concentration of 1 μM, can efficiently promote the proliferation and differentiation of hPDLSCs both in vitro and in vivo.

scholarly journals Methylprednisolone Protects SAP and Pancreatitis-Associated ALI in Mice by Inhibiting NLRP3 Inflammasome Through NF-κB

2020 ◽  
Author(s):  
Chuan-jiang Liu ◽  
Qiang Fu ◽  
Wenjing Zhou ◽  
Xu Zhang ◽  
Rui Chen ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Nlrp3 Inflammasome ◽  
Antioxidant Properties ◽  
Underlying Mechanism ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Expression Levels ◽  
Tnf Α

Abstract Background: Methylprednisolone (MP) is a synthetic corticosteroid with potent anti-inflammatory and antioxidant properties used as therapy for a variety of diseases. The underlying mechanism of MP to reduce acute pancreatitis still needs to be elucidated.Methods: Twenty-four male C57BL/6 mice (6-8 weeks) were used to establish SAP mouse model by administering an intraperitoneal injection of Cae and LPS. Amylase expression levels of serum and PLF were measured with an amylase assay kit. The concentrations of IL-1β and TNF-α in the serum and PLF were detected by ELISA. The level of pancreatic and lung tissue damage and inflammation was assessed by H&E staining and immunofluorescence staining. Western blot and qPCR were used to detect the expression levels of NLRP3, IL-1β and TNF-αin vivo and in vitro.Results: In this study, we found MP, used in the early phase of SAP, decreased the levels of IL-1β and TNF-α in serum and peritoneal lavage fluids (PLF), reduced the level of serum amylase and the expression of MPO in lung tissue, attenuated the pathological injury of the pancreas and lungs in a dose-dependent manner. The expression of NLRP3 and IL-1β in pancreas and lungs was down-regulated significantly depending on the MP concentration. In vitro, MP reduced the levels of IL-1β and TNF-α by down-regulating the expression of NLRP3, IL-1β and p-NF-κB in isolated peritoneal macrophages. Conclusion: MP can attenuate the injury of pancreas and lungs, and the inflammatory response in SAP mice by down-regulating the activation of NF-κB and the NLRP3 inflammasome.

scholarly journals Piglets produced by transfer of embryos obtained by in vitro fertilization of oocytes matured in vitro with thymosin: A case report

2020 ◽  
Vol 76 (03) ◽  
pp. 6356-2020 ◽  
Author(s):  
KATARZYNA PONIEDZIAŁEK-KEMPNY ◽  
BARBARA GAJDA ◽  
IWONA RAJSKA ◽  
LECHOSŁAW GAJDA ◽  
ZDZISŁAW SMORĄG
Keyword(s):  
Case Report ◽  
In Vitro Fertilization ◽  
Control Group ◽  
First Birth ◽  
Vitro Fertilization ◽  
Research Material ◽  
Preliminary Study ◽  
Experimental Group

The aim of the study was to examine the in vivo viability of in vitro-produced (IVP) porcine embryos obtained from oocytes matured with thymosin. The research material for this study consisted of immature pig oocytes obtained from ovaries after slaughter and ejaculated semen obtained from one boar. The immature oocytes were cultured in vitro until the metaphase II stage in a medium supplemented with thymosin (TMS). The presumptive zygotes obtained were cultured in vitro for 4-40 hours. The presumptive zygotes and 2-4-cell embryos were evaluated in vivo after transferring them to synchronized recipients. After the transfer of embryos from the experimental group into 2 recipients (50 embryos into each gilt) and the transfer of 50 embryos from the control group into 1 recipient, both gilts that had received embryos obtained by in vitro fertilization of oocytes matured with TMS became pregnant and delivered a total of 16 live piglets. After the transfer of embryos from the control group, no pregnancy was achieved. In conclusion, the results of our preliminary study suggest that the maturation of pig oocytes with thymosin supports the in vivo survival of in vitro produced embryos. It is important to note, that this was the first birth of piglets obtained after transfer of IVP embryos in Poland.

scholarly journals The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Dong Chen ◽  
Yaqin Wang ◽  
Feiya Yang ◽  
Adili Keranmu ◽  
Qingxin Zhao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Bioinformatic Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

2020 ◽  
pp. 15-34
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Tissue Regeneration ◽  
Real Time Pcr ◽  
Molecular Level ◽  
Northern Blotting ◽  
Tissue Constructs ◽  
Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

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