Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions

2015 ◽  
Vol 2015 (3) ◽  
pp. pdb.prot081018 ◽  
Author(s):  
Donald C. Rio
Keyword(s):  
Northern Blots ◽  
Agarose Gels ◽  
Filter Hybridization

Increased lung endothelin-1 production in rats with idiopathic pulmonary hypertension

1992 ◽  
Vol 262 (5) ◽  
pp. L614-L620 ◽  
Author(s):  
T. J. Stelzner ◽  
R. F. O'Brien ◽  
M. Yanagisawa ◽  
T. Sakurai ◽  
K. Sato ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Mrna Expression ◽  
Chronic Hypoxia ◽  
Sprague Dawley ◽  
Northern Blots ◽  
Endothelin 1 ◽  
Right Ventricular Size ◽  
Filter Hybridization ◽  
Idiopathic Pulmonary Hypertension

The role of endogenous circulating or locally produced endothelin-1 (ET-1) in pulmonary hypertensive states is unknown. To investigate this we measured ET-1 levels and preproendothelin-1 (prepro-ET-1) mRNA expression at various ages in control Sprague-Dawley (SDR) rats and in fawn-hooded rats (FHR), a strain which develops idiopathic pulmonary hypertension. Although serum ET-1 levels were similar in SDR and FHR, we found twofold increases in FHR whole lung homogenate ET-1 levels by radioimmunoassay. Coexisting threefold increases in preproET-1 mRNA expression were found in FHR lungs by densitometric analysis of Northern blots and by filter hybridization, suggesting the increase in lung ET-1 was due to enhanced intrapulmonary production of the peptide. To test whether the increase in lung preproET-1 mRNA was primary or secondary to established pulmonary hypertension, we compared preproET-1 mRNA expression prior to development of pulmonary hypertension in fetal (19 day gestation) and neonatal (5 day old) FHR and SDR. Despite similar right ventricular size in SDR and FHR, preproET-1 mRNA was already elevated in neonatal FHR lungs. Furthermore, we found no increase in lung preproET-1 mRNA or ET-1 levels in adult SDR with an equivalent degree of pulmonary hypertension due to chronic hypoxia, implying that the increases in ET-1 production in FHR were not a common consequence of all pulmonary hypertensive states. The functional significance of these observations remains unclear but raises the possibility of a role for ET-1 in the pathophysiology of pulmonary hypertension in the FHR.

Tomato DNA contains no detectable regions complementary to potato spindle tuber viroid as assayed by solution and filter hybridization

Virology ◽  
1980 ◽  
Vol 104 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Milton Zaitlin ◽  
C.L. Niblett ◽  
Elizabeth Dickson ◽  
R.B. Goldberg
Keyword(s):  
Potato Spindle Tuber Viroid ◽  
Filter Hybridization

scholarly journals Rapid transfer of DNA from agarose gels to nylon membranes

1985 ◽  
Vol 13 (20) ◽  
pp. 7207-7221 ◽  
Author(s):  
Ken C. Reed ◽  
David A. Mann
Keyword(s):  
Agarose Gels ◽  
Rapid Transfer

scholarly journals Extreme PCR: Efficient and Specific DNA Amplification in 15–60 Seconds

2015 ◽  
Vol 61 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Jared S Farrar ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
High Resolution Melting ◽  
Dna Amplification ◽  
Temperature Cycling ◽  
High Yield ◽  
Temperature Cycle ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Agarose Gels

Abstract BACKGROUND PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown. METHODS We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4–2.0 s at annealing/extension temperatures of 62 °C–76 °C and denaturation temperatures of 85 °C–92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring. RESULTS Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15–60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any “hot start” technique. Amplification efficiencies were 91.7%–95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles. CONCLUSIONS The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4–2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.

scholarly journals Differential mucin gene expression in human pancreatic and colon cancer cells

1991 ◽  
Vol 276 (3) ◽  
pp. 599-605 ◽  
Author(s):  
S Yonezawa ◽  
J C Byrd ◽  
R Dahiya ◽  
J J L Ho ◽  
J R Gum ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Human Pancreatic Cancer ◽  
Northern Blots ◽  
Western Blots ◽  
Pancreatic Cancer Cells

The purpose of this study was to determine the quantity and nature of the mucins synthesized and secreted by four different pancreatic cancer cell lines. Well- to moderately-differentiated SW1990 and CAPAN-2 human pancreatic cancer cells were found to produce more high-Mr glycoprotein (HMG) than less-differentiated MIA PaCa-2 and PANC-1 cells. Most of the labelled HMG was secreted within 24 h. The results of chemical and enzymic degradation, ion-exchange chromatography and density-gradient centrifugation indicated that the HMG in SW1990 and CAPAN-2 cells has the properties expected for mucins, whereas much of the HMG in MIA PaCa-2 and PANC-1 cells may not be mucin, but proteoglycan. These results are consistent with immunoblots and Northern blots showing the presence of apomucin and apomucin mRNA in SW1990 and CAPAN-2 cells, but not in MIA PaCa-2 and PANC-1 cells. The Western blots and Northern blots also show that SW1990 and CAPAN-2 cells, like breast cancer cells, have the mammary-type apomucin and mRNA coded by the MUC1 gene, but lack the intestinal type apomucin and mRNA coded by the MUC2 gene. In contrast, the colon cancer cell lines tested in culture express apomucin and mRNA coded by MUC2 but not by MUC1.

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