The Click-iT® Nascent RNA Capture Kit, uses our proprietary Click-iT® chemistry to capture with high efficiency and sensitivity newly minted RNAs. By the Click-iT® approach studies on high resolution analysis of gene regulation can be performed along with RNA regulation, RNA stability, RNA synthesis, RNA decay and degradation, transcriptional regulation, delta⁄delta C(t), nuclear run on⁄run off and more. Ethylene uridine (EU) ribonucleotide homologs containing an alkyne reactive group are fed to cells or tissue. Once incorporated, cells are lysed and the RNA isolated. A biotin azide is 'clicked on’ and then strepavidin magnetic beads are used to capture the newly synthesized pool of RNA. The captured RNAs are then amplified and the resultant cDNAs are used on any number of post-capture methodologies. We have validated it for arrays (both high and low density), sequencing on SOLiD™ and either SYBR® or TaqMan® based qPCR based delta CT analysis. As few as 25,000 cells are needed for the analysis of newly induced transcripts. The kit allows for 40 conditions for labeling and capture that can then be divided into 400 separate analytical assayss. With a price of around dollar an assy this is perfect for researchers in academia and industrial settings.
The EU reagent at the recommend concentration of 200 µM is well tolerated by cells. In an array screen of over 32,000 genes we could detect only the most minor changes in 12 compared to a control vehicle. Furthermore at initial feeding concentration 5x of what we recommend (200 µM vs 1.0 mM) there were no effects on a panel of housekeeping genes, or on the cells ability to proliferate normally. Sensitivity and reliability was confirmed in a low density array (TLDA®) of apoptotic genes. The captured nascent pool accurately found all the previously characterized apoptotic (stauroporine induced) transcripts, without any detectable change in sequence information.
*Discovery not just recovery.* The last decades Genomic efforts revealed that only 2% of the human encodes protein, leaving 98% of the human genome as the Unknome. Discovering the purpose and expression patterns of this vast reservoir of unknown sequence will be markedly aided in this decade’s transcriptomics by this new approach.
*Ability to study RNA Stability. * For others the ability to discover the stability of mRNA’s, without the need for radioactivity will be the key feature offered here. In the past transcript half lives were derived with radioactive nucleotides in traditional nuclear run and run off experiments. Cumbersome and dangerous, this approach is seen a steep decline – now these critical values can be derived simply, safely and reliably.
*Want to know what’s new?* Incorporate the Click-iT Line of metabolic analogs to label the new stuff! Without radioactivity or difficult to use haptens. Find what’s new in DNA, RNA, protein, sugars, ptms and more.
*Click-iT® Technology - One Reaction, Endless Possibilities!*