Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani

Background Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is one of the most important pathogens of rice (Oryza sativa L.) that causes severe yield losses in all rice-growing regions. Sclerotia, formed from the aggregation of hyphae, are important structures in the life cycles of R. solani and contain a large quantity of polysaccharides, lipids, proteins and pigments. In order to extract high-quality total RNA from the sclerotia of R. solani, five methods, including E.Z.N.A.™ Fungal RNA Kit, sodium dodecyl sulfate (SDS)-sodium borate, SDS-polyvinylpyrrolidone (PVP), guanidinium thiocyanate (GTC) and modified Trizol, were compared in this study. Results The electrophoresis results showed that it failed to extract total RNA from the sclerotia using modified Trizol method, whereas it could extract total RNA from the sclerotia using other four methods. Further experiments confirmed that the total RNA extracted using SDS-sodium borate, SDS-PVP and E.Z.N.A.™ Fungal RNA Kit methods could be used for RT-PCR of the specific amplification of GAPDH gene fragments, and that extracted using GTC method did not fulfill the requirement for above-mentioned RT-PCR experiment. Conclusion It is concluded that SDS-sodium borate and SDS-PVP methods were the better ones for the extraction of high-quality total RNA that could be used for future gene cloning and expression studies, whereas E.Z.N.A.™ Fungal RNA Kit was not taken into consideration when deal with a large quantity of samples because it is expensive and relatively low yield.

Chronic maternal dietary iodine deficiency but not thiocyanate feeding affects maternal reproduction and postnatal performance of the rat.

Iodine deficiency disorders affect reproductive performance in the afflicted populations. Environmental iodine deficiency (ID) and goitrogens are important in their aetiology. We observed earlier that chronic maternal dietary ID but not goitrogen feeding altered the blood-brain barrier nutrient transport in adult rats. Whether similar differences exist in their effects on reproduction of dams and postnatal performance of the offspring has been assessed. Inbred, female, weaning WNIN rats were rendered hypothyroid by feeding for 8-12 weeks, a low iodine test diet or a control diet with added potassium thiocyanate (KSCN) (@ 25 mg/rat/day). Following mating with control males, they continued on their respective diets till their pups were weaned. Indices of reproductive performance such as percentage of conception, mortality of dams during pregnancy and parturition, litter size, and survival of pups till weaning were affected markedly by ID but not thiocyanate feeding. Neither ID nor thiocyanate feeding from conception or parturition affected their reproductive performance. Nevertheless, postnatal weight gain of pups was less in all the three ID groups but not thiocyanate fed dams. Rehabilitation of chronically ID pregnant dams from conception or parturition did not improve their pregnancy weight gain, litter size or birth weight of pups but decreased abortion and mortality of mothers during pregnancy and parturition. Rehabilitation improved the pups' postnatal weight gain but the effect was only moderate. Based on the results of the present study it may be suggested that maternal ID but not thiocyanate feeding affects reproductive performance and postnatal performance of their offspring.

Rapid extraction of DNA from diverse soils by guanidine thiocyanate method.

Molecular methods are being frequently used for the study of soil microbial communities as majority of naturally occurring microbial populations are non-culturable. In the present study, we describe a protocol of DNA extraction from diverse soils using a combination of heat, enzyme (lysozyme) and guanidine thiocyanate. The efficacy of the procedure was evaluated in terms of yield, purity and duration of extraction. The protocol was effective for neutral, acidic as well as alkaline soils (pH range 4.5-8.5). The extracted soil DNA was observed with negligible shearing on agarose gel and the time taken for restriction digestion was very less. Further, the DNA extracted was almost completely devoid of contaminants and pure enough which could be used for PCR amplification and Southern hybridization.

Extractive spectrophotometric determination of some skeletal muscle relaxant drugs through ion-pair formation with molybdenum [V] and thiocyanate

A spectrophotometric procedure was developed for the determination of orphenadrine citrate [I], tizanidine hydrochloride [II] and pancuronium bromide [III]. The method was based on ion-pair complex formation with molybdenum [V] thiocyanate complex in an acidic medium. Dichloromethane was used for the extraction of the orange red ion-pair and its absorbance was measured at 470 nm. The molar absorptivities were 1.34 x 104, 1.19 x 104 and 4.35 x 104 1 mol-1 cm-1 for I, II and III, respectively. The ion-pair was stable for more than one day at room temperature. The proposed method was applied successfully for the analysis of samples in bulk powder and in dosage forms with recoveries in the range of 99-101%, 99.8-101.4% and 98.9-101.2%, respectively

Determinación de tiocianato en saliva y orina y su validación como parámetro de fumado / Determination of thiocyanate in saliva and urine and its validation as a smoking parameter

Se analizó una muestra de cien individuos voluntarios, cincuenta fumadores y cincuenta no fumadores, a los cuales se les determinó la concentración de tiocianato en saliva y orina; con el fin de verificar si este parámetro es adecuado para discriminar entre fumadores y no fumadores de tabaco. Se empleó un método colorimétrico sencillo, de bajo costo; el cual se basa en la reacción del tiocianato con Fe+3 formando un producto coloreado con máxima absorbancia a 460 nm. El método presenta coeficientes de variación día a día de 1,8 y 9,8 por ciento para concentraciones de tiocianato de 2.564 y 40 µmol/L respectivamente. El intervalo analítico es de 0 a 3.000 µmol/L y se obtuvo una recuperación promedio de 99 por ciento. Los promedios de la concentración de tiocianato en orina fueron de 3,8 y 96,6 230mol/L y en saliva de 646 y 2.521 µmol/L para no fumadores y fumadores respectivamente, estas diferencias son significativas (p < 0,0001). Los niveles de decisión, para diferenciar personas fumadoras de no fumadoras, son de 25 y 1.500 230mol/l, para muestras de orina y saliva respectivamente. Usando estos valores se obtuvo una eficiencia diagnóstica del 91 por ciento en orina y 89 por ciento en saliva

Interaction of guanidine hydrochloride and guanidine thiocyanate with wheat germ lipase.

Effect of two classical and potent denaturants, guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuHSCN) on purified wheat germ lipase has been studied. Lipase was found to be active only up to 5 M GuHCl and 1.5 M GuHSCN. The extent of interaction was determined by the measurement of apparent partial specific volume of the enzyme in presence of these two denaturants. While the preferential interaction parameter (zeta 3) has values of 0.08 +/- 0.02 and 0.14 +/- 0.03 g/g, the interaction parameter (delta m3/delta m2)T,mu 1, mu3 has values of 35 +/- 9 and 50 +/- 10 mole/mole for GuHCl and GuHSCN, respectively. The number of denaturant molecules bound to the enzyme, A3, obtained experimentally were 0.486 +/- 0.020 and 0.348 +/- 0.020 g/g and the calculated values were 0.459 +/- 0.023 and 0.567 +/- 0.030 g/g for 6 M GuHCl and 3 M GuHSCN, respectively. The volume change occurring upon denaturation results in -420 +/- 42 and -462 +/- 84 ml/mole in 6 M GuHCl and 3 M GuHSCN, respectively. The denaturation is accompanied by exposure of hydrophobic groups to the bulk solvent as confirmed by fluorescence emission measurements of the enzyme. The Tm measurements indicated a control value of 56 +/- 1 degree C. In presence of 6 M GuHCl/3 M GuHSCN, the value was 42 +/- 1 degree C. These results explain the retention of lipase activity even at 5 M GuHCl from a mechanistic point of view.