Microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the sensitivity, accuracy, specificity, and reproducibility are major challenges for this technology. Cross-hybridization, combination with splice variants, is a prime source for the discrepancies in differential gene expression calls among various microarray platforms. Removing (either from production or downstream bioinformatic analysis) and/or redesigning the microarray probes prone to cross-hybridization is a reasonable strategy to increase the hybridization specificity and hence, the accuracy of the microarray measurements.
Get tips on using In Situ Cell Death Detection Kit, TMR red to perform TUNEL assay cell type - HEK293 human embryonic kidney cells
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.
Microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the sensitivity, accuracy, specificity, and reproducibility are major challenges for this technology. Cross-hybridization, combination with splice variants, is a prime source for the discrepancies in differential gene expression calls among various microarray platforms. Removing (either from production or downstream bioinformatic analysis) and/or redesigning the microarray probes prone to cross-hybridization is a reasonable strategy to increase the hybridization specificity and hence, the accuracy of the microarray measurements.
Get tips on using GeneChip® Human Genome U133 Plus 2.0 Array to perform Microarray Human - Precision cut lung slices Expression array
DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.
DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.
When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.
When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.
When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.
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