The process of RNA extraction from bacteria, in general, involves an RNA-protective, effective lysis of bacterial cell wall (which may pose difficulties). EDTA promotes loss of outer membrane to provide lysozyme with access to peptidoglycan. Another common method for cell wall lysis is mechanical disruption using a homogenizer (applied for gram-positive bacteria and some strains of gram-negative bacteria). Following lysis, it is necessary to disrupt protein-nucleic acid interactions, which can be achieved by adding sodium dodecyl sulfate (SDS). Next step involves using phenol-chloroform-isoamyl alcohol extraction, where RNA can be obtained from the bottom organic phase, the top phase consists of DNA and the interphase contains proteins. Isoamyl alcohol is an inert and optional addition to this mixture and is added as an anti-foaming reagent to reduce the interphase. Following RNA extraction, the samples should be checked for its quality by gel electrophoresis (23S and 16S rRNAs and 5s rRNA and tRNA bands) or UV spectrophotometric or fluorescence methods.
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An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.
An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.
An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.
ELISA is the most commonly used method of detecting and quantifying the concentration of an antigen in an unknown sample. During the experiment, If you get a weak signal, then make sure reagents are at room temperature before starting the assay. Try increasing incubation times to ensure maximal antibody binding and amplify the signal. Secondly, if you get values above 0 in the negative control indicates a high background signal. Try to consider reducing your antibody concentration and prevent non-specific binding of antibodies by using affinity-purified antibody and suitable blocking buffers. To avoid high well to well variation, do not stack plates during incubation, no bubbles in the plate and wash wells thoroughly to avoid variation.
Get tips on using SurePrint G3 Human CGH Microarray Kit, 4x180K to perform Microarray Comperative genomic hybridization - Human BT474
Get tips on using SurePrint G3 Human CGH Microarray Kit, 4x180K to perform Microarray Comperative genomic hybridization - Human SKBR3
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