RESUMO
Dimethyl sulfide (DMS) is the major biogenic volatile sulfur compound in surface seawater. Good quality DMS data with high temporal and spatial resolution are desirable for understanding reduced sulfur biogeochemistry. Here we present a fully automated and novel "microslug" gas-liquid segmented flow-chemiluminescence (MSSF-CL) based method for the continuous in-situ measurement of DMS in natural waters. Samples were collected into a flow tank and DMS transferred from the aqueous phase to the gas phase using a vario-directional coiled flow, in which microvolume liquid and gas slugs were interspersed. The separated DMS was reacted with ozone in a reaction cell for CL detection. The analytical process was automated, with a sample throughput of 6.6 h-1. Using MSSF for DMS separation was more effective and easily integrated with CL detection compared with the commonly used bubbling approach. Key parameters of the proposed method were investigated. The linear range for the method was 0.05-500 nM (R2 = 0.9984) and the limit of detection (3 x S/N) was 0.015 nM, which is comparable to the commonly used gas chromatography (GC) method and sensitive enough for direct DMS measurement in typical aquatic environments. Reproducibility and recovery were assessed by spiking natural water samples (river, lake, reservoir and pond) with different concentrations of DMS (10, 20 and 50 nM), giving relative standard deviations (RSDs) ≤1.75% (n = 5) and recoveries of 94.4-107.8%. This fully automated system is reagent free, easy to assemble, simple to use, portable (weight ~5.1 kg) and can be left in the field for several hours of unattended operation. The instrumentation can provide high quality DMS data for natural waters with an environmentally relevant temporal resolution of ~9 min.
RESUMO
Cloning of differentially expressed genes is one of the hottest topics in biology.It is very important in cloning genes correlated with phenotype and diseases at molecular level.Here we utilized a simple and rapid PCR-based protocol to detect and isolate cDNA fragments from differentially expressed genes of IL-6-induccd and IL-6-uninduced U937 cells in tWO easy steps.To generate cDNAs from most mRNAs,the first step was reverse transcription using three fully degenerated 6-mer oligonucleotides as primers.The second step was PGR amplification of internal regions of the cDNAs with two or three longer primers with arbitrary but defined sequences.The PCR amplification was repeated on the same cDNA templates(first step) with different sets of primers.DNA fragments were easily displayed by 2% agarose gel electrophoresis and then the differential recovered fragments were used directly in cloning,sequencing,and RNA reverse Northern blot analysis.In this study,seven differential ESTs are obtained;two Sequences not found in GenBank,are novel ESTs.They were proved to be differentially expressed genes related with IL-6 effect by reverse Northern hybridization.
RESUMO
Cloning of differentially expressed genes is one of the hottest topics in biology.It is very important in cloning genes correlated with phenotype and diseases at molecular level.Here we utilized a simple and rapid PCR-based protocol to detect and isolate cDNA fragments from differentially expressed genes of IL-6-induccd and IL-6-uninduced U937 cells in tWO easy steps.To generate cDNAs from most mRNAs,the first step was reverse transcription using three fully degenerated 6-mer oligonucleotides as primers.The second step was PGR amplification of internal regions of the cDNAs with two or three longer primers with arbitrary but defined sequences.The PCR amplification was repeated on the same cDNA templates(first step) with different sets of primers.DNA fragments were easily displayed by 2% agarose gel electrophoresis and then the differential recovered fragments were used directly in cloning,sequencing,and RNA reverse Northern blot analysis.In this study,seven differential ESTs are obtained;two Sequences not found in GenBank,are novel ESTs.They were proved to be differentially expressed genes related with IL-6 effect by reverse Northern hybridization.
