Quantification of germ-cell-specific gene products by fluorescence real-time RT-PCR - a new technique for fast reliable molecular-diagnostic classification of spermatogenesis disorders

Technical innovations in brain tumour diagnostic and therapy have led to significant improvements of patient outcome and recurrence free interval. The use of technical devices such as surgical microscopes as well as neuronavigational systems have helped localising tumours as much as fluorescent agents, such as 5-aminolaevulinic acid, have helped visualizing pathologically altered tissue. Nonetheless, intraoperative instantaneous frozen sections and histological diagnosis remain the only method of gaining certainty of the nature of the resected tissue. This technique is time consuming and does not provide close-to-real-time information. In gastroenterology, confocal endoscopy closed the gap between tissue resection and histological examination, providing an almost real-time histological diagnosis. The potential of this technique using a confocal laser endoscope EndoMAG1 by Karl Storz Company was evaluated by our group on pig brains, tumour tissue cell cultures, and fresh human tumour specimen. Here, the authors report for the first time on the results of applying this new technique and provide first confocal endoscopic images of various brain and tumour structures. In all, the technique harbours a very promising potential to provide almost real-time intraoperative diagnosis, but further studies are needed to provide evidence for the technique’s potential.

Download Full-text

Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

JOURNAL FOR CLINICAL AND DIAGNOSTIC RESEARCH ◽

10.7860/jcdr/2021/48726.14977 ◽

2021 ◽

Author(s):

Rajeev Kumar Jain ◽

Nagaraj Perumal ◽

Rakesh Shrivastava ◽

Kamlesh Kumar Ahirwar ◽

Jaya Lalwani ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Internal Control ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Specific Gene ◽

Rt Pcr ◽

Chain Reaction ◽

Gene Target ◽

Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

Download Full-text

A New Technique to Measure Real Time Static and Dynamic Gasket Stresses

10.4271/910205 ◽

1991 ◽

Cited By ~ 2

Author(s):

Daniel E. Czernik ◽

Frank L. Miszczak

Keyword(s):

Real Time ◽

New Technique ◽

A New Technique

Download Full-text

A beacon in the cytoplasm: Tracking translation of single mRNAs

The Journal of Cell Biology ◽

10.1083/jcb.201608075 ◽

2016 ◽

Vol 214 (6) ◽

pp. 649-652 ◽

Cited By ~ 1

Author(s):

Hema V. Pingali ◽

Angela K. Hilliker

Keyword(s):

Real Time ◽

New Technique ◽

Real Time Monitoring ◽

Protein Levels ◽

Cell Biol ◽

A New Technique ◽

Control Protein

Translation is carefully regulated to control protein levels and allow quick responses to changes in the environment. Certain questions about translation in vivo have been unattainable until now. In this issue, Pichon et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201605024) describe a new technique to allow real-time monitoring of translation on single mRNAs.

Download Full-text

410. Gene expression analysis in endometriotic lesions using laser capture microdissection

Reproduction Fertility and Development ◽

10.1071/srb08abs410 ◽

2008 ◽

Vol 20 (9) ◽

pp. 90

Author(s):

L. Fu ◽

J. E. Girling ◽

P. A. W. Rogers

Keyword(s):

Gene Expression ◽

Real Time ◽

Laser Capture Microdissection ◽

Stromal Cells ◽

Expression Profiles ◽

Specific Gene ◽

Rt Pcr ◽

Normal Endometrium ◽

Rna Quality ◽

Laser Capture

Previous studies examining gene expression profiles in normal endometrium and endometriotic lesions have used RNA extracted from whole tissue samples. Results from these studies can be difficult to interpret as they reflect expression averaged across several different cell types that may be functionally quite different. The aim of this study was to establish laser capture microdissection (LCM) as a technique to examine gene expression in stromal and epithelial cells from normal and ectopic endometrium. We hypothesised that genes associated with inflammation would be elevated in cells from endometriotic lesions. Full thickness uterine samples were collected during abdominal hysterectomy from normal cycling premenopausal women. Endometriotic lesions were collected during abdominal laparoscopy. Samples were either frozen in OCT or stored in RNAlater for 12 h before freezing. Tissues were immunostained with an antibody against CD10 to identify ectopic endometrial stromal cells before LCM. Endometrial epithelial and stromal cells were collected using the PALM MicroLaser System. RNA quality was accessed using Experion. TGFβ1, MMP1, αSMA, SMAD2 and NFκB mRNA was analysed using real-time RT–PCR. Of the endometriotic samples stored in OCT (n = 58), only 14% (n = 8) had visible endometrial glands. Of these, only 37% (n = 3) had RNA of an acceptable quality for further analysis. However, RNA quality and quantity were dramatically improved in 3 of 5 samples collected in RNAlater. In preliminary studies, expression of TGFβ1 and αSMA mRNA was elevated in endometriotic lesions in comparison to the normal endometrium, whereas NFκB expression did not change. We have shown that RNAlater solution is useful to preserve RNA quality for small clinical endometriotic samples and that immuno-guided LCM-generated homogenous cell populations coupled with real-time RT–PCR can provide valuable insights into cell and disease-specific gene expression in endometriotic lesions.

Download Full-text