Simple method for extracting RNA from cultured cells and tissue with guanidine salts

1993 ◽  
Vol 39 (7) ◽  
pp. 1408-1411 ◽  
Author(s):  
R Zolfaghari ◽  
X Chen ◽  
E A Fisher
Keyword(s):  
Cultured Cells ◽  
Guanidine Hydrochloride ◽  
Placental Tissue ◽  
Animal Origin ◽  
Clinical Samples ◽  
Simple Method ◽  
Simple Protocol ◽  
Human Placental Tissue ◽  
Polymerase Chain

Abstract We have developed a simple protocol for isolating RNA from both cell culture and tissue from human and animal sources, using guanidine thiocyanate and guanidine hydrochloride, but no organic solvents. The protocol reproducibly yielded 15 to 25 micrograms of high-quality RNA per 10(6) cells of human and animal origin and 1 to 1.1 mg of RNA per gram of human placental tissue. The RNA so obtained was ribonuclease-free and not contaminated by DNA. It was suitable for reverse transcription-polymerase chain reaction, Northern blot analysis, and in vitro expression of proteins. Thus, the molecular assessment of both research and clinical samples can be readily and reliably initiated by the application of this protocol.

Studies on Diagnosis of Bovine Brucellosis by Immunocyto-chemistry and Immunohistochemistry

2021 ◽  
Author(s):  
K.S. Lakshmikanth ◽  
N.S. Sharma ◽  
D. Pathak ◽  
Paviter Kaur
Keyword(s):  
Biochemical Analysis ◽  
Placental Tissue ◽  
Diagnostic Techniques ◽  
Clinical Samples ◽  
Bovine Brucellosis ◽  
Reproductive Disorders ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Polymerase Chain ◽  
Positive Results

Background: Brucellosis is a major threat to livestock economy and an important zoonotic disease. A rapid and accurate diagnosis is a necessity to curb the spread and progress of the disease. The current study aimed to evaluate sensitivity of Immunocytochemistry and Immunohistochemistry methods for detection of Brucella spp.Methods: A total of 50 samples comprising of fetal stomach content, vaginal discharges and placenta were collected from cattle and buffaloes suffering from abortions and other reproductive disorders in and around Ludhiana, Punjab during the period 2017-2018. All the samples were processed for isolation and confirmed with biochemical analysis and Polymerase chain reaction (PCR). The isolates obtained and 43 clinical samples excluding placental samples were subjected to Immunocytochemistry (ICC). Immunohistochemistry (ICH) was performed on placental samples.Result: A total of four isolates were recovered from the screened samples. The four isolates also yielded positive results in Immunocytochemistry. Among the 43 clinical samples screened by Immunocytochemistry, five were positive, however only 3 isolates were recovered on isolation. A total of seven placental tissue samples were processed and subjected to immunohistochemistry. Of the three placental samples positive by immunohistochemistry, only one sample was isolated on culture. The results suggest that both immunocytochemistry and immunohistochemistry are sensitive diagnostic techniques in comparison to isolation.

scholarly journals Potential Significance of Circular RNA in Human Placental Tissue for Patients with Preeclampsia

2016 ◽  
Vol 39 (4) ◽  
pp. 1380-1390 ◽  
Author(s):  
Yating Qian ◽  
Yuanqing Lu ◽  
Can Rui ◽  
Yujia Qian ◽  
Manhong Cai ◽  
...  
Keyword(s):  
Operating Characteristic ◽  
Placental Tissue ◽  
Circular Rna ◽  
Circular Rnas ◽  
Potential Significance ◽  
Human Placental Tissue ◽  
Polymerase Chain ◽  
Mirna Sponges ◽  
Basic Content ◽  
Experimental Group

Aims: This study aimed to identify the different expression of circular RNAs (circRNAs) in the placental tissues of pregnant women with preeclampsia (PE) and to provide a new avenue of research regarding the pathological mechanisms of PE. Methods: In this study, we collected 40 placental tissues from PE patients and 35 placental tissues from gestational age-matched patients who gave premature birth. Arraystar circRNA Microarray Technology (KANGCHEN, Shanghai, China) was used to analyze the differential expression of circRNAs. According to the basic content of circRNAs in the two groups and their fold changes and due to the practicability of the designed divergent primers of each candidate circRNA, we selected three up-regulated circRNAs, hsa_circRNA_100782, hsa_circRNA_102682 and hsa_circRNA_104820, to validate the data. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was utilized to estimate the Ct values in both groups. We further evaluated the differences with a paired t-test and a receiver operating characteristic (ROC) curve. Results: Many circRNAs were found to be differentially expressed in PE placental tissues versus their controls; of these, 143 circRNAs were up-regulated and 158 were down-regulated. The expression levels of hsa_circRNA_100782 (p < 0.05), hsa_circRNA_102682 (p < 0.05), and hsa_circRNA_104820 (p < 0.0001) were validated as significantly up-regulated in the experimental group compared with the controls. Finally, we performed a literature comparison to forecast the possible mechanisms of circRNA function during PE. Conclusion: circRNA expression significantly differed in placental PE tissues compared with controls. According to the circRNA microarray results and the existing papers, circRNAs may contribute to the pathogenesis of PE by acting as miRNA sponges; this possibility requires additional investigation in future studies.

EFFECT OF IONIC ENVIRONMENT ON THE RELEASE OF HUMAN PLACENTAL LACTOGEN IN VITRO

1976 ◽  
Vol 69 (3) ◽  
pp. 349-358 ◽  
Author(s):  
V. J. CHOY ◽  
W. B. WATKINS
Keyword(s):  
Placental Tissue ◽  
Placental Lactogen ◽  
Human Placental Lactogen ◽  
Short Term ◽  
Graded Response ◽  
High K ◽  
Human Placental Tissue ◽  
High Concentrations ◽  
Free Media

SUMMARY Short-term incubation of human placental tissue in Krebs–Ringer bicarbonate buffered media with various concentrations of K+ and Ca2+ showed a graded response in human placental lactogen (HPL) release at different Ca2+ concentrations, but no effect at increased K+ concentration. Media with high Ca2+ caused an inhibition of release, while Ca2+-free media caused a stimulation in HPL release. High concentrations of Mg2+ inhibited release minimally, while Ba2+ had no effect. There was no change in HPL release when Na+ concentration was increased. La3+-Locke's solution markedly inhibited release of HPL but the significance of this effect is unknown. These results suggest that Ca2+ is not required for HPL secretion from placental tissue. It seems that HPL secretion in vitro does not follow the usual pattern where a physiological stimulus or high K+ concentration causes inward movement of calcium which couples stimulation to secretion.

TRANSFORMATION IN VITRO OF [4-14C]DEHYDROEPIANDROSTERONE TO 7-OXYGENATED DERIVATIVES BY NORMAL EARLY HUMAN PLACENTAL TISSUE

1971 ◽  
Vol 50 (2) ◽  
pp. 301-306 ◽  
Author(s):  
Y. W. MIRHOM ◽  
F. E. SZONTÁGH
Keyword(s):  
Placental Tissue ◽  
Human Placental Tissue ◽  
Early Human ◽  
Human Placentae

SUMMARY Samples from three early human placentae were incubated with [4-14C]-dehydroepiandrosterone (DHA). It was found that [4-14C]DHA was transformed into 7-oxygenated derivatives in yields decreasing in the order: 7-oxo-DHA > 7β-hydroxy-DHA > 7α-hydroxy-DHA. These products were only slowly transformed into other derivatives.

scholarly journals Adrenomedullin gene expression in human placental tIssue and leukocytes: a potential marker of severe tIssue hypoxia in neonates with birth asphyxia

2002 ◽  
pp. 711-716 ◽  
Author(s):  
R Trollmann ◽  
E Schoof ◽  
E Beinder ◽  
D Wenzel ◽  
W Rascher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Perinatal Asphyxia ◽  
Newborn Infants ◽  
Birth Asphyxia ◽  
Placental Tissue ◽  
Tissue Hypoxia ◽  
Mrna Levels ◽  
Potential Marker ◽  
Human Placental Tissue

OBJECTIVE: The aim of the present study was to investigate the role of adrenomedullin (ADM) as a hypoxia-inducible marker of clinically relevant tIssue hypoxia in acute birth asphyxia of term newborn infants. METHODS: For this purpose, ADM mRNA was determined in human placental tIssue of 20 term pregnancies complicated by birth asphyxia (pH and base deficit values, clinical score). In addition, ADM mRNA was measured in leukocytes of the asphyxiated newborn infants during the first 12 h of life (n=12). Controls were available from ten healthy term pregnancies. In vitro, hypoxia-inducible expression of ADM mRNA was evaluated in human choriocarcinoma cells (BeWo) and human leukocytes exposed to hypoxia (1% O(2)) for 1-24 h. mRNA levels were measured by TaqMan real-time PCR. RESULTS: In vitro, ADM mRNA related to porphobilinogen deaminase (PBGD) mRNA levels significantly increased in response to hypoxia within a period of 4 h in leukocytes and 12 h in BeWo cells. In human placental tIssue, significantly higher levels of ADM/PBGD mRNA were present in asphyxiated newborn infants with severe hypoxic-ischemic encephalopathy (HIE) (n=5) compared with patients with mild or no HIE (n=15). Increased levels of ADM/PBGD mRNA levels were found during the first hours of life in leukocytes of neonates with severe HIE compared with controls. CONCLUSIONS: Our results indicate an upregulation of ADM gene expression in human placenta and leukocytes in clinically relevant hypoxic-ischemic birth complications and suggest ADM gene expression as a promising marker for severe complications due to perinatal asphyxia such as HIE.

In vitro growth-stimulatory property of pigeon milk

1993 ◽  
Vol 71 (5-6) ◽  
pp. 303-307 ◽  
Author(s):  
L. Bharathi ◽  
K. B. Shenoy ◽  
M. Mojamdar ◽  
S. N. Hegde
Keyword(s):  
Cell Lines ◽  
Cho Cells ◽  
Cultured Cells ◽  
Guanidine Hydrochloride ◽  
Chinese Hamster ◽  
Growth Stimulation ◽  
Relative Mass ◽  
Good Growth ◽  
In Vitro Growth

Five cell lines were employed to test the growth-stimulating property of pigeon milk in vitro. All the cell lines except A431 showed good growth response to crude homogenates of pigeon milk. Enhancement of DNA synthesis in quiescent Chinese hamster ovary (CHO) cells by pigeon milk was dose dependent up to a concentration of 1%. In vitro growth stimulation by 1% pigeon milk was approximately equal to that by 2% foetal bovine serum (FBS) when CHO cells were used, growth stimulation of Vero cells by 1% pigeon milk was roughly three times of that by 2% FBS. In contrast, 1% pigeon milk was only half as active as 2% FBS on NIH/3T3 cells and five times less active than 2% FBS on human foetal lung fibroblast cells. After dialysis using a relative mass (Mr) cutoff of 3500, the pigeon milk mitogenic activity was retained in the dialyzed solution, although it decreased by 40–60% when dialyzed with Mr cutoffs of 8000 and 12 000 – 14 000. The growth-stimulating activity of pigeon milk was resistant to heat, acid, alkali, and the action of urea, guanidine hydrochloride, dithiothreitol, and trypsin. We suggest that pigeon milk is a new source of growth factor(s) capable of stimulating in vitro the growth of many mammalian cell types.Key words: pigeon milk, growth stimulation, cultured cells.

scholarly journals A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257770
Author(s):  
Kazuyo Watanabe ◽  
Mikio Yoshiyama ◽  
Gaku Akiduki ◽  
Kakeru Yokoi ◽  
Hiroko Hoshida ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Honey Bee ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Plasmid Vector ◽  
Simple Method

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace’s insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

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