NFkB is a ubiquitous transcription factor that plays a key role in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. Incorrect regulation of NFkB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development. When NFkB is activated, it is dissociated from its inhibitor IkB and moves from the cytoplasm to the nucleus, where it binds to target DNA elements and positively regulates the transcription of genes involved in immune and inflammatory responses, cell growth control, and apoptosis. To get a better overview of gene expression of NFkB regulated genes, Signosis has developed a cDNA Plate Array which couples its CLTM cDNA synthesis kit which allows reverse transcription directly from cell lysates without RNA preparation. This plate-based assays allows profiling the expression of 20+ NFkB-regulated genes directly in cell lysates. This assay provides quantitative data, allowing researchers to screen multiple samples, as well as high sensitivity, allowing researchers to use less sample.
Unlike conventional reverse transcription which needs total RNA, Signosis has developed a CL(TM) cDNA synthesis-based plate array. It can synthesize cDNA probes through reverse transcription directly in cell lysates without RNA preparation. The synthesized cDNA probes are then applied for a plate array hybridization. Targeted genes are specifically captured onto individual wells on a plate through a pre-coated gene-specific oligonucleotide. The captured cDNAs are further detected with streptavidin-HRP. Luminescence is reported as relative light units (RLUs) on a microplate luminometer. The expression level of genes is directly proportional to the luminescent intensity.
Human NFkB-regulated cDNA plate array analysis. HeLa cells were treated with (red bars) or without (light blue) 10ng/ml TNF for 30 min. Cells were collected for profiling 23 genes with the cDNA plate array assay (top). Time course of IL-6 induction to TNFa treatment in HeLa cells was monitored with the cDNA plate array assay.
DNA can be synthesized directly in cell lysates without RNA preparation. In addition, the detection is as simple as ELISA
Highly Sensitive Detection
Detected mRNA molecules are converted into multiple biotin-labeled cDNAs for sensitive detection
The difference of two or more samples in gene expression can be quantitatively analyzed