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 TRIzol Reagent to perform RNA isolation / purification Cells - immortalized HNSCC
Get tips on using RNeasy Mini Kit to perform RNA isolation / purification Cells - immortalized HNSCC
Get tips on using RNeasy Plus Mini Kit to perform RNA isolation / purification Cells - immortalized HNSCC
Get tips on using pMHL_2002-bfloGFPa1 to perform Protein Expression Eukaryotic cells - T. pseudonana IbpA DR2 antigen from Histophilus somni
Get tips on using Mouse Sonic Hedgehog/Shh DuoSet ELISA to perform ELISA Mouse - ShhN
Get tips on using Cyclin E1 (HE12) Mouse mAb #4129 to perform Western blotting Cyclin E
Get tips on using Mouse Heme Oxygenase 1 ELISA Kit (ab204524) to perform ELISA Mouse - HO-1
Fill out your contact details and receive price quotes in your Inbox
Outsource experiment