A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.
2-mercaptoethanol (2-ME), alongside polyvinylpyrrolidone is commonly used in plant DNA extractions to deal with polyphenols, which could interfere with extraction and downstream applications. 2-ME is also commonly used to denature proteins and nucleases, especially RNAses. On the contrary, we found that the presence of 2-ME in lysis buffer interfered with DNA extraction from 12 strains of freshwater microalgae, resulting in DNA with poor integrity. We also found that the TNES-urea buffer, commonly used for preservation and DNA extraction from fish, appears as effective as the SDS and CTAB buffer for some microalgae strains. Results from our study suggests that the inclusion of 2-ME in DNA extraction protocols may be detrimental for isolation of good quality DNA from freshwater microalgae, and therefore recommend eliminating it or testing varying concentrations of 2-ME when developing species-specific extraction protocols for microalgae.
We successfully developed an in-house, competitive enzyme immunoassay to measure advanced glycosylation end-products (AGE) in serum. The assay involved coating microtitre wells with AGE-BSA at 8 micrograms/ml for 4 hours, followed by overnight incubation of 20 microliters sample (prediluted at 1:6) with 80 microliters antiserum (1:8000). HRP-labelled goat anti-rabbit was used as the second antibody and 3,5',5,5'-tetramethylbenzidine dihydrochloride as the substrate. Incubation was carried out at 4 degrees C. As suggested in an earlier study, we standardised the AGE units against normal human serum (NHS). Thus, one AGE unit was defined as the inhibition that resulted when the 1:6 diluted NHS was assayed. Mean (+/- SD) AGE level in normal subjects (n = 37) was significantly lower than in diabetes subjects with microalbuminuria (n = 57) (6.0 +/- 0.7 versus 10.2 +/- 4.7 units/ml, p = 0.0001). With the availability of in-house assay and by standardising the AGE unit with the other laboratories, more studies could be undertaken and results compared, and possibly, further elucidate the roles of AGE in the pathogenesis of diabetic complications.
The merger of nanotechnology and combination chemotherapy has shown notable promise in the therapy of resistant tumors. The latest scientific attention encompasses the engagement of anticancer drugs in combination with small interfering (si)RNAs, such as VEGF, XLAP, PGP, MRP-1, BCL-2 and cMyc, to name but a few. siRNAs have shown immense promise to knockout drug resistance genes as well as to recover the sensitivity of resistant tumors to anticancer therapy. The nanotechnology approach could also protect siRNA against RNAse degradation as well as prevent off-target effects. In this article, we discuss the approaches that have been used to deliver of siRNA in combination with chemotherapeutic drugs to treat resistant tumors. We also discuss the stipulations that must be considered in formulating a nanotechnology-assisted siRNA-drug cancer therapy.