Relative Expression Levels of SERCA in Panulirus argus and Homarus americanas tissues using Quantitative Real Time PCR

Background Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants. Methods In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. Results The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants.

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Gene-Related Strain Variation of Staphylococcus aureus for Homologous Resistance Response to Acid Stress

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-070 ◽

2014 ◽

Vol 77 (10) ◽

pp. 1794-1798 ◽

Cited By ~ 2

Author(s):

SOOMIN LEE ◽

SOOYEON AHN ◽

HEEYOUNG LEE ◽

WON-IL KIM ◽

HWANG-YONG KIM ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Real Time ◽

Real Time Pcr ◽

Acid Resistance ◽

Transcriptional Analysis ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Resistance Response ◽

Expression Levels ◽

Acid Shock

This study investigated the effect of adaptation of Staphylococcus aureus strains to the acidic condition of tomato in response to environmental stresses, such as heat and acid. S. aureus ATCC 13565, ATCC 14458, ATCC 23235, ATCC 27664, and NCCP10826 habituated in tomato extract at 35°C for 24 h were inoculated in tryptic soy broth. The culture suspensions were then subjected to heat challenge or acid challenge at 60°C and pH 3.0, respectively, for 60 min. In addition, transcriptional analysis using quantitative real-time PCR was performed to evaluate the expression level of acid-shock genes, such as clpB, zwf, nuoF, and gnd, from five S. aureus strains after the acid habituation of strains in tomato at 35°C for 15 min and 60 min in comparison with that of the nonhabituated strains. In comparison with the nonhabituated strains, the five tomato-habituated S. aureus strains did not show cross protection to heat, but tomato-habituated S. aureus ATCC 23235 showed acid resistance. In quantitative real-time-PCR analysis, the relative expression levels of acid-shock genes (clpB, zwf, nuoF, and gnd) were increased the most in S. aureus ATCC 23235 after 60 min of tomato habituation, but there was little difference in the expression levels among the five S. aureus strains after 15 min of tomato habituation. These results indicate that the variation of acid resistance of S. aureus is related to the expression of acid-shock genes during acid habituation.

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In Acute Promyelocytic Leukemia (APL) with Multiple Relapses Additional Prognostic Information Is Provided by FLT3 and Telomerase (hTERT) Quantitative Real-Time PCR.

Blood ◽

10.1182/blood.v104.11.4408.4408 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4408-4408

Author(s):

Paolo Bernasconi ◽

Silvia Calatroni ◽

Barbara Rocca ◽

Paola Maria Cavigliano ◽

Carlo Castagnola ◽

...

Keyword(s):

Real Time ◽

Induction Chemotherapy ◽

Real Time Pcr ◽

Clinical Relapse ◽

Htert Expression ◽

Quantitative Real Time Pcr ◽

Molecular Remission ◽

Expression Levels ◽

Flt3 Expression ◽

Multiple Relapses

Abstract The present study, carried out in two APL with multiple relapses, was aimed at determining 1) which correlation does exist between FLT3/hTERT expression levels and PML-RARA results; 2) whether high FLT3 and hTERT expression levels might be predictive of relapse; 3) whether FLT3 expression is better than FLT3 Internal Tandem Duplication (ITD) for evaluating disease outcome. Relative quantifications of FLT3/hTERT transcripts were performed by real-time PCR using SybrGreen I. For FLT3 calibration total RNA from a normal subject was used, for hTERT total RNA from K562 cells. In both cases the ΔΔCt method was used for quantification. On clinical diagnosis one patient with a WBC of 124.0x109/L and a PML-RARA fusion at PML BCR1 presented the FLT3/hTERT genes highly expressed. On qualitative PCR the patient also showed the ITD of FLT3. He was treated with the AIDA protocol and succeeded in achieving a haematological but not molecular remission. During CR FLT3/hTERT expression remained high and the ITD was never detected. Fourteen months later when on first clinical relapse FLT3 expression abruptly increased, the ITD reappeared, hTERT levels were still high. A re-induction chemotherapy induced a second haematological but not molecular remission lasting five months. FLT3 as well as hTERT expression levels became similar to those of the control, and FLT3 ITD disappeared. A progressive increase of FLT3 expression and an abrupt increase of hTERT expression preceded the second relapse which was accompanied by the reappearance of the ITD. After re-induction chemotherapy FLT3/hTERT expression dropped down to values of the control. A third CR was obtained but the patient remained PML/RARA and Flt3 ITD positive and soon after died of a CNS relapse. The other patient was treated in another Centre and came to our observation in haematological CR. At that time he was PML-RARA negative with high FLT3/hTERT expression. Eight months later he was still in clinical but not molecular CR having a PML-RARA fusion at BCR3, high FLT3/hTERT expression levels and presenting FLT3 ITD. One month later when clinical relapse occurred FLT3 expression levels were unchanged, hTERT expression dropped down to normal values and FLT3 ITD was still present. A re-induction chemotherapy induced a second CR with alternatively positive and negative PML-RARA results, high FLT3 and low hTERT expression levels. The patient underwent an allogeneic bone marrow transplant from an unrelated donor but five months later he relapsed for the second time with an abrupt rise of hTERT expression that preceded a quick increase of FLT3 expression. A third clinical but not molecular CR was achieved after chemotherapy, but the patient remained PML-RARA positive with a normal FLT3/hTERT expression. Two months later a rapid increase of hTERT expression preceded that of FLT3 and the occurrence of the third relapse. In conclusion i) increased FLT3 and hTERT levels during CR are associated with alternative positive/negative PML-RARA results on nested RT PCR and are always predictive of pending relapse; ii) on disease recurrence a marked elevation of hTERT expression often preceded that of FLT3; iii) quantitative real-time PCR of the FLT3 gene was more effective in predicting disease outcome than the ITD, this last being discovered only when FLT3 expression was already high.

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Lack of correlation between in silico projection of function and quantitative real-time PCR-determined gene expression levels in colon tissue

Pharmacogenomics and Personalized Medicine ◽

10.2147/pgpm.s49199 ◽

2013 ◽

pp. 99

Author(s):

Susan Kadlubar ◽

Rosalind Penney ◽

Abbie Lundgreen ◽

Aiwei Yao-Borengassar ◽

Vineetha Koroth-Edavana ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

In Silico ◽

Quantitative Real Time Pcr ◽

Colon Tissue ◽

Expression Levels ◽

Gene Expression Levels

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NF2 Expression Levels of Gastrointestinal Stromal Tumors: A Quantitative Real-Time PCR Study

Tumori Journal ◽

10.1177/030089160809400417 ◽

2008 ◽

Vol 94 (4) ◽

pp. 551-555 ◽

Cited By ~ 4

Author(s):

Antonio Taddei ◽

Francesca Castiglione ◽

Duccio Rossi Degl'Innocenti ◽

Anna Maria Buccoliero ◽

Francesca Garbini ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Gastrointestinal Stromal Tumors ◽

Quantitative Real Time Pcr ◽

Expression Levels ◽

Stromal Tumors

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Simultaneous Quantitative Detection of Relevant Biomarkers in Breast Cancer by Quantitative Real-Time PCR

The International Journal of Biological Markers ◽

10.1177/172460080602100105 ◽

2006 ◽

Vol 21 (1) ◽

pp. 30-39 ◽

Cited By ~ 12

Author(s):

M. Labuhn ◽

V. Vuaroqueaux ◽

F. Fina ◽

A. Schaller ◽

I. Nanni-Metellus ◽

...

Keyword(s):

Breast Cancer ◽

Mrna Expression ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Detection ◽

Mrna Levels ◽

Quantitative Real Time Pcr ◽

Expression Levels ◽

Qrt Pcr ◽

Mrna Expression Levels

The assessment of ERα, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERα, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERα, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

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Gualou Guizhi Granule Protects against OGD/R-Induced Injury by Inhibiting Cell Pyroptosis via the PI3K/Akt Signaling Pathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/6613572 ◽

2021 ◽

Vol 2021 ◽

pp. 1-14

Author(s):

Yuqin Zhang ◽

Hongyun Wang ◽

Huang Li ◽

Lihong Nan ◽

Wei Xu ◽

...

Keyword(s):

Ischemic Stroke ◽

Western Blot ◽

Real Time ◽

Real Time Pcr ◽

Akt Signaling ◽

Akt Pathway ◽

Pcr Analysis ◽

Akt Signaling Pathway ◽

Quantitative Real Time Pcr ◽

Expression Levels

Pyroptosis is a proinflammatory form of regulated cell death that plays an important role in ischemic stroke. Gualou Guizhi granule (GLGZG) is a classic prescription that has been shown to exert neuroprotective effects against cerebral ischemia reperfusion injury. In the present study, we examined the involvement of pyroptosis and its associated mechanism in protecting nerve function. Methods. Primary neurons were exposed to oxygen-glucose deprivation and reperfusion (OGD/R) conditions in the presence or absence of GLGZG. Cellular viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay. The number of apoptoic cells was detected by NeuN and NSE protein expression. The expression levels of the pyroptosis markers, namely, NOD-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, interleukin-18 (IL-18), and IL-1β were determined by quantitative real-time PCR analysis, western blot, and ELISA analyses as appropriate. Moreover, the expression levels of the PI3K/Akt pathway key proteins were determined by quantitative real-time PCR analysis and western blot assays. To determine the PI3K/Akt pathway involvement in GLGZG-mediated neuroprotection, the PI3K inhibitor LY294002 (LY, 10 μM) was added. The expression levels of NeuN, Akt, and p-Akt were evaluated. Results. It was found that GLGZG could inhibit OGD/R-induced cell apoptosis, increase neuronal cell viability, decrease the production of IL-18 and IL-1β, and downregulate the expression levels of pyroptosis markers (NLRP3, ASC, and caspase-1). Furthermore, GLGZG could modulate the PI3K/Akt signaling pathway. Pharmacological inhibition of the PI3K pathway not only abrogated the effects of GLGZG on Akt but also neutralized its prosurvival and antipyroptotic actions. Conclusions. The findings indicated that GLGZG pretreatment effectively reduced OGD/R-induced injury by inhibiting cell pyroptosis and activating the PI3K/Akt pathway. These data provide important evidence for the therapeutic applications of this regimen in ischemic stroke.

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