Invitrogen™ Click-iT™ Nascent RNA Capture Kit, for Gene Expression Analysis

Invitrogen™ Click-iT™ Nascent RNA Capture Kit, for Gene Expression Analysis

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.

For Use With (Equipment):7000 System, 7200 System, 7300 System, 7500 Fast System, 7500 System, 7900HT Fast System, 7900HT System, StepOne™, Fast Mode, StepOne™, Standard Mode, StepOnePlus™, Fast Mode, StepOnePlus™, Standard Mode, Veriti Thermal Cycler, Agilent 2100 Bioanalyzer, SOLiD™

Contents

• 5-ethynyl Uridine (EU, Component A): 5 mg
• Click-iT® EU buffer (Component B): 1 mL
• Biotin azide (PEG4 carboxamide-6-azidohexanyl biotin) (Component C): 340 μg
• Copper (II) Sulfate (CuSO4) (Component D): 200 μL
• Click-iT® reaction buffer additive 1 (Component E): 400 mg
• Click-iT® reaction buffer additive 2 (Component F): 100 μL
• Click-iT® RNA binding buffer (Component G): 2 × 1 mL
• Dynabeads® MyOne™ Streptavidin T1 (Component H): 500 μL
• Click-iT® reaction wash buffer 1 (Component I): 35 mL
• Click-iT® reaction wash buffer 2 (Component J): 35 mL