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.
Get tips on using Iscove’s Modified Dulbecco’s Medium to perform Stem cell culture media hMegakaryoblastic cells
Get tips on using CelLytic™ MT Cell Lysis Reagent to perform Protein isolation Mammalian cells - HeLa
Get tips on using Phusion Site-Directed Mutagenesis Kit to perform Site Directed Mutagenesis (SDM) Human - Deletion MCF-7 sodium channel β1 subunit
Get tips on using Cell Lysis Buffer (10X) to perform Protein isolation Mammalian cells - HepG2
Get tips on using GeneArt™ Site-Directed Mutagenesis System to perform Site Directed Mutagenesis (SDM) Human - Point mutation THP-1 IL-33
Get tips on using Q5® Site-Directed Mutagenesis Kit to perform Site Directed Mutagenesis (SDM) Human - Point mutation MDA-MB-231 NANOG
Get tips on using SNAI 1 siRNA and shRNA Plasmids (h) to perform siRNA / miRNA gene silencing Human - MDA-MB-468 SNAI 1
Get tips on using SNAI 1 siRNA and shRNA Plasmids (h) to perform siRNA / miRNA gene silencing Human - MDA-MB-231 SNAI 1
Fill out your contact details and receive price quotes in your Inbox
Outsource experiment