Site Directed Mutagenesis (SDM) Monkey Deletion COS-7

Get tips on using Gibco™ DMEM, high glucose, GlutaMAX™ Supplement to perform 3D Cell Culture Media Mouse small intestinal organoids

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - primary rat aortic smooth muscle cells

Get tips on using jetPEI® DNA transfection, HTS application to perform DNA transfection Mammalian cells - Primary cells Rat aortic smooth muscle cells (rASMC)

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - primary human pulmonary artery smooth muscle cells

Get tips on using FlashTag™ Biotin HSR RNA Labeling Kits to perform Microarray RNA amplification & Labeling - Rat primary vascular smooth muscle cells Biotin

Get tips on using X-tremeGENE™ HP DNA Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Human aortic smooth muscle cells (HOSMC)

Get tips on using LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells to perform Live / Dead assay mammalian cells - rat aortic smooth muscle cells

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.

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.

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.