RNA isolation / purification Tissue Human

Get tips on using MicroRNA isolation kit to perform RNA isolation / purification Yeast - Saccharomyces cerevisiae

Get tips on using TriPure Isolation Reagent to perform RNA isolation / purification Cells - immortalized PC-12

Get tips on using TriPure Isolation Reagent to perform RNA isolation / purification Cells - immortalized RIN-5F

Get tips on using TriPure Isolation Reagent to perform RNA isolation / purification Cells - immortalized KG-1

Get tips on using Total Exosome RNA & Protein Isolation Kit to perform Protein isolation Mammalian cells - HeLa

Get tips on using Naive Pan T Cell Isolation Kit, human to perform Cell Isolation Naive Pan T cell

Get tips on using AquaRNA Kit to perform RNA isolation / purification Cells - primary human mesenchymal stem cells

Get tips on using miRNeasy Mini kit to perform RNA isolation / purification Cells - primary human cardiac fibroblasts

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.