Isolation of RNA from cell lines and cervical cytology specimens stored in BD SurePath™ preservative fluid and downstream detection of housekeeping gene and HPV E6 expression using real time RT-PCR

Studies examining gene expression with RT-PCR typically normalize their mRNA data to a constitutively expressed housekeeping gene. The validity of a particular housekeeping gene must be determined for each experimental intervention. We examined the expression of various housekeeping genes following an acute bout of endurance (END) or resistance (RES) exercise. Twenty-four healthy subjects performed either a interval-type cycle ergometry workout to exhaustion (∼75 min; END) or 300 single-leg eccentric contractions (RES). Muscle biopsies were taken before exercise and 3 h and 48 h following exercise. Real-time RT-PCR was performed on β-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β2-microglobulin (β2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at ∼65% of V̇o2 max, and we examined a fifth housekeeping gene, 28S rRNA, and reexamined β2M, from muscle biopsy samples taken immediately postexercise. We showed that CYC increased 48 h following both END and RES exercise (3- and 5-fold, respectively; P < 0.01), and 28S rRNA increased immediately following END exercise (2-fold; P = 0.02). β-Actin trended toward an increase following END exercise (1.85-fold collapsed across time; P = 0.13), and GAPDH trended toward a small yet robust increase at 3 h following RES exercise (1.4-fold; P = 0.067). In contrast, β2M was not altered at any time point postexercise. We conclude that β2M and β-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas β2M and GAPDH are the most stably expressed following END exercise.

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Increased Expression of the Pro-Protein Convertase Furin Predicts Decreased Survival in Ovarian Cancer

Analytical Cellular Pathology ◽

10.1155/2007/930321 ◽

2007 ◽

Vol 29 (4) ◽

pp. 289-299

Author(s):

Robert E. Page ◽

Andrés J. P. Klein-Szanto ◽

Samuel Litwin ◽

Emmanuelle Nicolas ◽

Raid Al-Jumaily ◽

...

Keyword(s):

Ovarian Cancer ◽

Tumor Progression ◽

Real Time ◽

Cell Lines ◽

Cancer Cell ◽

Real Time Pcr ◽

Ovarian Tumor ◽

Cancer Cell Lines ◽

Rt Pcr ◽

Primary Tumors

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

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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.

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Real-Time RT-PCR Quantification of Human Telomerase Reverse Transcriptase Splice Variants in Tumor Cell Lines and Non–Small Cell Lung Cancer

Clinical Chemistry ◽

10.1373/clinchem.2006.073015 ◽

2007 ◽

Vol 53 (1) ◽

pp. 53-61 ◽

Cited By ~ 29

Author(s):

Eleni Mavrogiannou ◽

Areti Strati ◽

Aliki Stathopoulou ◽

Emily G Tsaroucha ◽

Loukas Kaklamanis ◽

...

Keyword(s):

Tumor Cell ◽

Real Time ◽

Cell Lines ◽

Splice Variant ◽

Splice Variants ◽

Telomerase Activity ◽

Tumor Cell Lines ◽

Rt Pcr ◽

Human Telomerase Reverse Transcriptase ◽

Tissue Samples

Abstract Background: We developed and validated a real-time reverse transcription (RT)–PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants (α+β+, α−β+, α+β−, α−β−) in tumor cell lines and non–small cell lung cancer (NSCLC). Methods: We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript–specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients. Results: In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The α+β− splice variant showed the highest expression and α−β+ and α−β− the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the α+β− variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by α+β+, with the α−β+ and α−β− splice variants having the lowest expression. In the NSCLC tumors, the α+β+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival. Conclusions: Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT α+β+ splice variant may be an independent negative prognostic factor for NSCLC patients.

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Effects of Acceleration Amplitude and Frequency of Mechanical Vibration on Cultured Osteoblasts

Volume 2: Biomedical and Biotechnology Engineering ◽

10.1115/imece2008-67221 ◽

2008 ◽

Author(s):

Tetsuo Shikata ◽

Toshihiko Shiraishi ◽

Shin Morishita ◽

Ryohei Takeuchi

Keyword(s):

Real Time ◽

Mechanical Stimulation ◽

Bone Cells ◽

Bone Matrix ◽

Mechanical Vibration ◽

Housekeeping Gene ◽

Rt Pcr ◽

Calcium Salts ◽

Acceleration Amplitude ◽

Gene Level

This paper describes the effects of the frequency and acceleration amplitude of mechanical vibration on osteoblasts, the bone cells that generate the bone matrix. Their cell proliferation and bone matrix generation were investigated when sinusoidal inertia force was applied to the cells. Bone formation is subject in vivo to mechanical stimulation. Although many researches for bone cells of osteoblastic lineage sensing and responding to mechanical stimulation have been reported mainly in the biochemical field, effects of mechanical stimulation on bone cells are not well understood. After the cells were cultured in culture plates in a CO2 incubator for one day and adhered on the cultured plane, vibrating groups of the culture plates were set on an aluminum plate attached to a exciter and cultured under sinusoidal excitation in another incubator separated from non-vibrating groups of the culture plates. Acceleration amplitude and frequency were set to several kinds of conditions. The time evolution of cell density was obtained by counting the number of cells with a hemocytometer. Calcium salts generated by the cells were observed by being stained with alizarin red S solution and their images were captured with a CCD camera. The vibrating groups for the cell proliferation and the calcium salts staining were sinusoidally excited for 24 hours a day during 28 days of culture. Gene expression of alkaline phosphatase (ALP) and runt-related gene 2 (Runx2) was measured by a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method. After the vibrating groups for the PCR were excited for 4 days, the total RNAs were extracted. After reverse transcription, real-time RT-PCR was performed. Gene expression for ALP, Runx2, and a housekeeping gene were determined simultaneously for each sample. ALP and Runx2 gene level in each sample was normalized to the measured housekeeping gene level. The following experimental results of sinusoidal excitation of osteoblasts have been shown: (a) Cell density decreased at 0.5 G with increasing frequency in the range from 12.5 to 1000 Hz and increased at 25 Hz with increasing acceleration amplitude from 0 to 0.5 G at 14 days of culture. (b) No calcium salts were observed in the non-vibrating group and the areas of calcium salts observed in the 0.5 G vibration group were larger than those in the 0.25 G group at 25 Hz at 21 days of culture. (c) The mRNA level of ALP at 0.5 G showed the peak at 50 Hz in the range from 12.5 to 1000 Hz and that at 50 Hz showed the peak at 0.5 G in the range from 0.25 to 1 G at 4 days of culture. In the case of Runx2, the same tendency was found. It has been shown that it is important to consider mechanical vibration as well as biochemical aspects in studies of the functional adaptation of cells to mechanical stimulation.

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A Novel Mechanism of ADAMTS13 Deficiency in Mice.

Blood ◽

10.1182/blood.v104.11.3668.3668 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3668-3668

Author(s):

Wenhua Zhou ◽

Lingli Dong ◽

Eric E. Bouhassira ◽

Han-Mou Tsai

Keyword(s):

Real Time ◽

Cell Lines ◽

Mammalian Cell ◽

Activity Level ◽

Full Length ◽

Mouse Strains ◽

Activity Levels ◽

Adamts13 Activity ◽

Rt Pcr ◽

Mammalian Cell Lines

Abstract Background. ADAMTS13, a circulating metalloprotease that cleaves conformationally altered von Willebrand factor (VWF), is critical for preventing microvascular thrombosis. Deficiency of the protease, due to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura. In the course of investigating the regulation of ADAMTS13, we noted that mice differed widely in their plasma ADAMTS13 activity levels. In order to understand the factors affecting plasma ADAMTS13 levels, we examined the molecular basis of ADAMTS13 variation in different strains of mice. Methods. Plasma ADAMTS13 activity level was determined by previously described SDS PAGE and immunoblotting. ADAMTS13 transcripts were analyzed by RT PCR, RACE, nucleotide sequencing, and real-time RT PCR. Plasmids containing the cDNA of mouse ADAMTS13 were constructed for transfection of mammalian cell lines. Results. The mouse strains FVB/NJ and 129X1/SvJ differed from C57BL/6J and DBA/2J by more than 10 folds in their plasma ADAMTS13 activity levels (3.09 +/− 0.45 vs 0.24 +/− 0.11 U/mL for FVB/NJ and C57BL/6J respectively, P < 0.0001). To determine the causes of the difference, we analyzed the ADAMTS13 transcripts by using RT PCR and RACE, which showed that the FVB/NJ mice contained the predicted full-length ADAMTS13 transcript with a domain structure similar to human ADAMTS13, while the C57BL/6J mice contained at least 4 isoforms: the full-length transcript, one internal splicing isoform, and two truncated forms that ended with an extraneous sequence homologous to the long-terminal repeat (LTR) of an retrotransposone of the IAP type. Comparison of the genomic sequences showed that the ADAMTS13 gene of C57BL/6J mice contained in its intron #23 an IAP-type retrotransposone sequence whose LTR sequence with a stop codon was included in the mouse transcripts. The IAP retrotransposone sequence, which contained one base substitution at the 5′-end 4-base repeat (tgtt>g) and was flanked at both ends by a 6-base repeat (cactag), was also present in the DBA/2J but not the 129X1/SvJ strains of mice. Real-time RT PCR showed that the FVB/NJ and C57BL/6J mice had similar levels of ADAMTS13 transcripts in the liver. Nevertheless in the C57BL/6J mice the IAP-truncated forms accounted for >90% of the ADAMTS13 transcripts. Expression of mouse ADAMTS13 cDNA in mammalian cell lines revealed that the both the full-length and the IAP-truncated forms of the ADAMTS13 protease were similar in VWF-cleaving activity. Conclusion. This study shows the presence of intragenic retrotransposone in the ADAMTS13 gene of some mouse strains. The presence of an IAP-retrotransposone within the ADAMTS13 gene of C57BL/6J mice affects the splicing of the ADAMTS13 transcripts, creating truncated forms of the protease that lack the last two TSP-1 and the CUB domains but remain proteolytically active in cleaving VWF. The lower plasma ADAMTS13 activity level of C57BL/6J may result from abnormal intravascular clearance of the protease or other post-secretory events.

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Chemokines expressed in melanoma metastases associated with T cell infiltration

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.8501 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 8501-8501 ◽

Cited By ~ 5

Author(s):

T. Gajewski ◽

Y. Meng ◽

H. Harlin

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Real Time ◽

Cell Lines ◽

Protein Array ◽

Rt Pcr ◽

Effector Phase ◽

Melanoma Metastases

8501 Background: Despite frequent induction of tumor antigen-specific T cells in melanoma patients following vaccination, tumor regressions remain rare. This observation prompted systematic analysis of the melanoma tumor microenvironment to identify factors that may influence the effector phase of the anti-tumor immune response. Methods: Gene expression profiling using the Affymetrix platform was performed on a series of melanoma metastases, melanoma cell lines, and primary melanocyte cell lines. Confirmatory assays were done by real-time RT-PCR, protein array, immunohistochemistry (IHC), and in vitro chemokine migration assays. Results: Non- supervised hierarchical clustering revealed 3 major subsets of tumors, with the main clustering based on differential expression of T cell-derived transcripts. The presence of CD8+ T cells was confirmed by IHC. Tumors that contained T cells uniquely expressed high levels of multiple chemokines. Protein array confirmed high expression of CCL2, CCL4, and CCL5; real-time RT-PCR additionally confirmed relatively high levels of CXCL9, CXCL10, and CCL3 transcripts. Transwell assays confirmed that each of these 6 chemokines recruited CD8+ effector cells in vitro. Conclusions: We have identified a set of 6 chemokines that likely regulates recruitment of activated T cells into melanoma metastases. Tumors that lack such chemokines might not be capable of supporting the effector phase of the anti-tumor immune response. We suggest that chemokine profiling of tumor sites should be performed in clinical trials of active immunotherapy. No significant financial relationships to disclose.

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