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Isolate It All: siRNA miRNA ,,, Total RNA Native Protein

species by Northern blot or by solution hybridization assay with the mirVana miRNA Detection Kit (Ambion). RNA probes were prepared by in vitro transcription (mRNA probes--MAXIscript Kit, Ambion) or by 5' end labeling (rRNA, miRNA and siRNA probes - mirVana Probe & Marker Kit, Ambion). Western blots were performed with 15 g of total protein and antibodies specific for GAPDH or Ku p70 (Ambion).


Here, GAPDH knockdown was triggered by electroporation of a GAPDH-specific siRNA into a primary human cell line (normal human umbilical vein endothelial cells, or HUVEC). A reduction of GAPDH expression was observed both at the mRNA and protein level in these cells, but not in HUVECs electroporated with Silencer Negative Control #1 siRNA. No variation in expression level was detected for the control mRNA (-actin), small RNAs (miR-16, 5S rRNA, 5.8S rRNA), or the control protein (Ku).


Compatible with Most Tissues
Although it was specifically designed to quickly process cultured cell samples, the mirVana PARIS Kit can also be used to isolate RNA and protein from most animal tissues. During the rapid homogenization step in Cell Disruption Buffer, RNA can be partially accessible to cellular ribonucleases. Thus, the procedure is not compatible with tissues that contain very high levels of these enzymes, such as pancreas or spleen. To test for potential loss of RNA integrity du
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