Long Rna Transfection Lipid Mediated Transfections, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins"
Article Title: Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins
Journal: PLoS ONE
Figure Legend Snippet: Long-RNA transfection yields ES-cell-level expression of reprogramming proteins in primary human fibroblasts. A. The transcribed strand of an Hbb -UTR-stabilized in vitro-transcription template encoding an arbitrary protein. The long arrow indicates the first transcribed base, and short arrows indicate restriction-enzyme cleavage sites. B. In vitro-transcribed RNA encoding reprogramming proteins. C. Western blots showing expression levels and lifetimes of Oct4, Sox2, Nanog, Lin28, and MyoD1 proteins in MRC-5 human fetal lung fibroblasts transfected with protein-encoding RNA, relative to levels in hES (H9) and rhabdomyosarcoma (Rh30) cells. β-actin was used as a loading control. Left panels: The amount of RNA per 50 µL electroporation volume was varied as indicated. Cells were lysed 6 hours after transfection. Right panels: Cells were transfected with 1 µg of RNA, and lysed at the indicated times. D. Expression and nuclear localization of Oct4, Sox2, Klf4, Utf1, Nanog, Lin28, and MyoD1 protein following long-RNA transfection. Cells were fixed and stained 6–12 hours after transfection. For each protein, identical camera settings and exposure times were used for the RNA-transfected and mock-transfected samples.
Techniques Used: Transfection, Expressing, In Vitro, Western Blot, Electroporation, Staining
Figure Legend Snippet: Innate immune suppression enables frequent long-RNA transfection. Combinatorial siRNA screening identifies siRNA cocktails that rescue cells from the innate immune response triggered by long-RNA transfection. A. Upregulation of innate immune genes following long-RNA transfection. MRC-5 fibroblasts were transfected with 0.4 µg of RNA per well of a 24-well plate using lipids. Expression of innate immune genes was measured by quantitative RT-PCR 24 hours after transfection. Gapdh was used as a loading control. Error bars indicate the standard deviation of replicate samples. B. Repeated long-RNA transfection causes cell death in human fibroblasts. MRC-5 fibroblasts were electroporated twice with 0.5 µg/50 µL of Lin28-encoding RNA at 48-hour intervals. Samples of cells transfected with RNA (black circles) and mock-transfected cells (gray squares) were trypsinized and counted at the indicated times. Data points and error bars indicate the mean and standard error of two independent experiments. Data points are connected for clarity. C. Combined knockdown of Ifnb1 , Eif2ak2 , and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. MRC-5 fibroblasts were transfected as in (B), but with the indicated siRNA on day 0, and 0.5 µg of Lin28-encoding RNA and additional siRNA on days 2 and 4 ( Table S1 ). Samples of cells were trypsinized and counted 24 hours after the second long-RNA transfection (day 5). Values indicate cell count relative to mock-transfected cells. † Standard error of replicate samples (n = 4). *p
Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation, Cell Counting
Figure Legend Snippet: Repeated long-RNA transfection yields sustained, high-level expression of active proteins that modulate downstream targets. A. Sustaining high levels of Lin28 protein expression by frequent transfection with Lin28-encoding RNA. MRC-5 fibroblasts pre-transfected with a cocktail of siRNAs targeting Ifnb1 , Eif2ak2 , Stat2 , and Tlr3 were transfected five times with 0.5 µg of Lin28-encoding RNA and additional siRNA at 48-hour intervals. Cells were lysed at the indicated times, and the amount of Lin28 protein was analyzed by western blot. β-actin was used as a loading control. B. Sustained expression of Lin28 downregulates its target, mature let7 miRNA. Transfections were conducted as in (A). Data points indicate mature let7a levels in cells transfected once (circles), twice (squares), three times (diamonds), four times (triangles), or five times (crosses), relative to the level in mock-transfected cells. A solid smoothed line connects data points corresponding to cells transfected once, and a dashed smoothed line connects data points corresponding to cells transfected five times (dark symbols). U47 RNA was used as a loading control. Error bars indicate the standard error of replicate samples. C. let7a downregulation is Lin28-specific. Cells were transfected as in (A), but with MyoD1-encoding RNA. Error bars indicate the standard error of replicate samples. D. Expression of MyoD1 protein in fibroblasts. Fibroblasts cultured for three days with or without 2.5 µM 5-aza-dC (AZA) were electroporated with 1 µg/50 µL of MyoD1-encoding RNA. Cells were lysed at the indicated times, and the amount of MyoD1 protein in each sample was analyzed by western blot. E. Expression of MyoD1 in fibroblasts activates its normally silent targets, Cdh15 and Des in a methylation-dependent manner. Cells were transfected as in (D), and expression of Cdh15 and Des was measured by RT-PCR at the indicated times (squares, mock-transfected cells; circles, RNA-transfected cells). F. Regulation of Hmga2 expression by reprogramming proteins. Hmga2 expression in fibroblasts transfected with RNA encoding the indicated protein was measured by RT-PCR 24 hours after transfection. Values are given relative to mock-transfected cells. Gapdh was used as a loading control. *p
Techniques Used: Transfection, Expressing, Western Blot, Cell Culture, Methylation, Reverse Transcription Polymerase Chain Reaction