siRNA / miRNA gene silencing Human HeLa EPAS-1

Get tips on using CONFIRM anti-Estrogen Receptor (ER) (SP1) Rabbit Monoclonal Primary Antibody to perform Immunohistochemistry Human - ER

Get tips on using DuoSet® ELISA Development Systems to perform ELISA (kit) IL-6, IL-1β, IL-8 and TNF-α - -NA- Human -NA-

Get tips on using DMEM/Ham's F-12 liquid medium w/o L-Glutamine to perform Stem cell culture media Human Tendon Stem/Pluripotence cells (TSPCs)

Get tips on using EasySep™ HLA B Cell Enrichment Kit to perform Cell Isolation HLA B Cell

Get tips on using EasySep™ HLA T Cell Enrichment Kit to perform Cell Isolation HLA T Cell

Get tips on using Agilent DNA 1000 Kit Bioanalyzer DNA Analysis Part Number:5067-1504 to perform Cell line authentication Human lung carcinoma cell line NCI-H1299

Get tips on using miRNeasy Serum/Plasma Advanced Kit (50) to perform RNA isolation / purification Tissue - Livestock Blood / Serum / Plasma / Buffy coat

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

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.