DNA isolation / purification Cells Immortalized cell lines

Get tips on using FuGENE® HD Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines H9C2

Get tips on using FuGENE® HD Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines Huh7

Get tips on using FuGENE® HD Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines HEK293

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines 293T

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines CHO

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines Huh7

Get tips on using ICAfectin®442 siRNA transfection to perform DNA transfection Mammalian cells - Immortalized cell lines COS7

Get tips on using ICAfectin®442 siRNA transfection to perform DNA transfection Mammalian cells - Immortalized cell lines HeLa

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines HepG2

The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.