siRNA / RNAi /miRNA transfection Human Cal 27 cells

Get tips on using ON-TARGETplus Mouse Cflar (12633) siRNA to perform siRNA / miRNA gene silencing Mouse - 3T3-SA Flip/CFLAR

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.

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.

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.

Get tips on using ON-TARGETplus Rat Dlc1 (58834) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - MTLn3 Dlc1

Get tips on using ON-TARGETplus Rat Rhoa (117273) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - MTLn3 Rhoa

Get tips on using ON-TARGETplus Rat Rhoc (295342) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - MTLn3 RhoC

Get tips on using ON-TARGETplus Rat Rhog (308875) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - MTLn3 RhoG

Get tips on using ON-TARGETplus Rat Trio (310192) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - MTLn3 Trio

Get tips on using ON-TARGETplus Rat Pak1 (29431) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - MTLn3 Pak1