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Invitrogen™ Click-iT™ Nascent RNA Capture Kit, for gene expression analysis
Description
Includes
Component A: 1 x 5mg 5-ethynyl uridine (EU); Component B: 1 x 1mL Click-iT EU buffer 2X concentrate; Component C: 1 x 340μg biotin azide (PEG4 carboxamide-6-azidohexanyl biotin); Component D: 1 x 200μL copper (II) sulfate (CuSO4) 25mM aqueous solution; Component E: 1 x 400mg Click-iT reaction buffer additive 1; Component F : 1 x 100μLl Click-iT reaction buffer additive 2; Component G: 2 x 1mL Click-iT RNA binding buffer (2X concentrate); Component H : 1 x 500μL Dynabeads™ MyOne™ Streptavidin T1; Component I: 1 x 35mL Click-iT reaction wash buffer 1; Component J: 1 x 35mLl Click-iT™ reaction wash buffer 2
The Click-iT Nascent RNA Capture Kit, uses 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 assays. 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 200μM is well tolerated by cells. In an array screen of over 32,000 genes, only the most minor changes in 12 compared to a control vehicle could be detected. Furthermore at initial feeding concentration 5x of what we recommend (200μM vs 1.0mM) 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.
- Less hazardous
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.
Click Chemistries, DNA and RNA Purification and Analysis, Gene Expression Analysis and Genotyping, Labeling Chemistry, Microarray Analysis, Nascent RNA Detection and Capture with Click Chemistry, Nucleic Acid Labeling and Oligo Synthesis, RNA Isolation for Expression Microarrays, RNA Labeling
Order Info
Shipping Condition: Wet ice
Specifications
Specifications
| Content And Storage | Component A 1 X 5 mg 5-ethynyl uridine (EU) Component B 1 X1 ml Click-iT™ EU buffer *2X concentrate* Component C 1 X 340 μg biotin azide (PEG4 carboxamide-6-azidohexanyl biotin) Component D 1 X 200 μl copper (II) sulfate (CuSO4) *25mM aqueous solution* Component E 1 X 400 mg Click-iT™ reaction buffer additive 1 Component F 1 X 100 μl Click-iT™ reaction buffer additive 2 Component G 2 X 1 ml Click-iT™ RNA binding buffer *2X concentrate* Component H 1 X 500 μl Dynabeads™ MyOne™ Streptavidin T1 Component I 1 X 35 ml Click-iT™ reaction wash buffer 1 Component J 1 X 35 ml Click-iT™ reaction wash buffer 2 Store at 4 degrees C |
| Concentration | 2X (EU Buffer & RNA Binding Buffer) |
| Format | Kit |
| 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™ |
| Isolation Technology | Magnetic Bead |
| Product Line | Click-iT |
| Product Type | Nascent RNA Capture Kit |
| Quantity | 1 kit |
| Shipping Condition | Wet Ice |
Frequently Asked Questions (FAQs)
Click reactions have several general characteristics: the reaction is efficient, no extreme temperatures or solvents are required, the reaction is complete within 30 minutes, the components of the reaction are bio-inert, and perhaps most importantly, no side reactions occur-the label and detection tags react selectively and specifically with one another. This final point is a key advantage of this powerful detection technique; it is possible to apply click chemistry-labeled molecules to complex biological samples and detect them with unprecedented sensitivity due to the extremely low background of the reaction.
Yes, cDNA produced at the end of the Click-iT Nascent RNA Capture Kit, for gene expression analysis (Cat. No. C10365) workflow (step 6.8 on page 11 of the Manual) can be used for next-generation sequencing (NGS) application.
Below are some references of the downstream NGS application using this kit:
Palozola, K. C., Donahue, G., & Zaret, K. S. (2021). EU-RNA-seq for in vivo labeling and high throughput sequencing of nascent transcripts. STAR protocols, 2(3), 100651.
Lu, X. M., Batugedara, G., Lee, M., Prudhomme, J., Bunnik, E. M., & Le Roch, K. G. (2017). Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum. Nucleic acids research, 45(13), 7825-7840.
For Research Use Only. Not for use in diagnostic procedures.