Product Introduction
This kit uses 5-ethynyluracil (EU) to label nascent RNA in living cells. The nascent RNA is then labeled with biotin through a click chemistry reaction. The nascent RNA is then specifically captured using streptavidin magnetic beads. The captured nascent RNA can then be used in subsequent applications such as qPCR and RNA sequencing. Combined with experimental timelines, this allows for quantitative analysis of newly generated RNA and the study of RNA transcription levels and degradation.
Features
Notes:
During the experiment, please wear a lab coat, mask, and disposable gloves, operate in a clean experimental area, and use RNase-free consumables;
The kit must be completely thawed and mixed before use. Keep the reagents on ice during use.
This product is for research use only.
Product ingredients:|
Reagent test kit |
Components |
FS-L1004-01 |
FS-L1004-02 |
FS-L1004-03 |
|
Kit 1 |
Click-Buffer-2 |
120 μL |
240 μL |
960 μL |
|
Protective agent |
600 μL |
1200 μL |
4800 μL |
|
|
Streptavidin magnetic beads |
240 μL |
480 μL |
1920 μL |
|
|
2×Wash Buffer |
1320 μL |
2640 μL |
10560 μL |
|
|
Solution A |
960 μL |
1920 μL |
7680 μL |
|
|
Solution B |
960 μL |
1920 μL |
7680 μL |
|
|
Kit 2 |
EU (1 M) |
100 μL |
200 μL |
800 μL |
|
Biotin |
36 μL |
72 μL |
288 μL |
|
|
copper sulfate |
36 μL |
72 μL |
288 μL |
|
|
Click-Buffer-1 |
72 μL |
144 μL |
576 μL |
Kit 1: Store at 2-8°C, valid for 12 months;
Kit 2: Store at -25~-15℃, valid for 12 months.
Steps:1.1 EU incubation of cell samples
When the cell density is 70-90% or the cell density meets the requirements of the experimental design, EU incubation is performed. Use complete culture medium to dilute EU. The final concentration of EU used in common cells is 1 mM and incubate for 2 hours. Alternatively, the final concentration of EU and incubation time can be adjusted according to literature reports. For the first experiment, a preliminary experiment can be performed according to the recommended EU dose and time; for example, after removing the culture medium of the cells in the T25 flask, add the prepared culture medium containing 1 mM EU to the culture flask and incubate for 2 hours. * Follow step 2 for subsequent experiments.
The following table shows the recommended EU doses and durations for testing:
|
EU concentration (mM) |
Incubation time (h) |
|
2 |
2 |
|
1 |
4 |
|
0.5 |
6-8 |
|
0.1 |
10-24 |
*Lung cancer cells (95D, H1573, H209, H226, H292, H460), liver cancer cells (HepG2, Huh7, SMMC-7721B, Hep3B, PLC/PRF/5, SK-HEP-1, BEL-7402, Mahlavu, Focus), breast cancer cells (MDA-MB-231, MDA-MB-468, HCC1937, SUM149, MCF- 7, T47D, CAMA-1, ZR-75-1, HCC1954, SK-BR-3, Hs578T, BT-594), ovarian cancer cells (A2780, SKOV3, ES2, HEY, CAOV3, OVCAR8, Anglne, UWB1.289), cervical cancer cells (HeLa, SiHa, CaSki, C-33A, ME-180, HT-3, MS751, DoTc2 Common cancer cells and non-cancerous cell lines include human ovarian cancer cells (VEGF, VEGF-κB, VEGF-κB, VEGF-κB), human gastric cancer cells (AGS, MKN-45, MKN-28, NCI-N87, SNU-1, HGC-27, NUGC-3, OCUM-1), colorectal cancer cells (HCT-116, HT-29, SW480, SW620, Caco-2, LoVo, DLD-1, COLO 205, RKO, LS 174T, LS 180, T84), renal cancer cells (786-O, Caki-1, Caki-2, ACHN, 769-P, A498, OS-RC-2), neuroblastoma cells (SH-SY5Y, SK-N-SH, IMR-32, LAN-1), osteoblasts (SJSA-1, TE-85), and neural cells (U87, NSC-13, U251, PC12).
1.2 EU incubation of animal tissues
According to the animal's body weight, the injection or gavage can be performed. EU can be prepared with PBS to a certain concentration at a dosage of 200-400 mg/kg and injected or gavaged. The specific dosage is related to the type of animal used, weight and method of use. You can refer to relevant literature. For example, when using for the first time, it is recommended to explore the concentration of EU or directly use a concentration of 200 mg/kg for testing. After 4 hours, * Alternatively, after an appropriate time determined for the specific experiment, quickly sacrifice the mouse and remove the desired tissue. Proceed with subsequent experiments according to step 2 in step 1.
*Experimentally verified in common animal models such as mice, rats, ground squirrels, and rabbits.
1.3 EU incubation of plant samples
Add EU for incubation according to the culture environment of the plant. The dosage can be determined by referring to relevant literature. For liquid culture, EU can be added to the culture medium to a final concentration of 0.5 mM and cultured for 6-8 hours. * You can extract RNA according to your own method. After extracting RNA, proceed to the second step of the click reaction follow-up experiment.
*Experimentally verified for hydroponic and soil-cultured ginseng, various flowers and grain crops, etc.
2.1 For cultured cell samples, remove the culture medium and lyse the cells with 1 ml of TRIZOL. Place the cells in an enzyme-free EP tube and let them stand at room temperature for 5 minutes. For plant and animal samples, obtain the corresponding tissue, wash the tissue 2-3 times with enzyme-free PBS, place the tissue in 1 ml of TRIZOL and grind it with a grinding instrument until no tissue fragments are visible. Place the tissue in an enzyme-free EP tube and let it stand at room temperature for 5 minutes.
2.2 Add 200 μL of chloroform, vortex to mix, and let stand at room temperature for 3 min;
2.3 Centrifuge at 14000g for 5 min, remove the supernatant, add an equal volume of isopropanol, and vortex to mix;
2.4 Place on ice for 10 min, centrifuge at 14,000 g for 20 min, and discard the supernatant.
2.5 Wash the pellet with 75% ethanol and centrifuge at 14,000 g for 5 min.
2.6 Repeat step 6;
2.7 Discard the supernatant. After the ethanol evaporates, add 50 μL of DEPC water to dissolve the RNA.
2.8 rRNA removal: The Qihengxing Ribosomal RNA Removal Kit is recommended for rRNA removal (this step is recommended. rRNA removal can improve the detection sensitivity of target RNA and reduce the sequencing amount).
3.1 Click reaction solution preparation
|
Element |
Volume/μL |
|
Biotin |
2.5 |
|
copper sulfate |
3 |
|
Click-Buffer-1 |
6 |
|
Click-Buffer-2 |
10 |
|
Protective agent |
50 |
|
PBS |
928.5 |
|
Total |
1000 |
3.2 Add 150 μL of click reaction solution to each sample and vortex to mix;
3.3 Place on ice for 30 min;
3.4 Add 1 ml of TRIZOL to the click reaction solution and vortex to mix.
3.5 Repeat the RNA extraction process (TRIZOL extraction).
4.1 Vortex the solution to fully suspend the streptavidin magnetic beads. Do not centrifuge to prevent the beads from settling.
4.2 Transfer 20 μL of resuspended magnetic beads to a new EP tube for each sample. For SMB magnetic beads required for multiple samples, wash them together, for example, by aliquoting the magnetic beads from 6 samples (120 μL) or 12 samples (240 μL) into one tube for washing. Add 20 μL of 2× Wash Buffer to each 20 μL of streptavidin magnetic beads, mix thoroughly, and rotate on a horizontal rotator at low speed for 1 minute. Place on a magnetic stand until the beads are adsorbed, and discard the supernatant.
4.3 After removing the EP tube containing the magnetic beads, add 40 μL of 2× Wash Buffer for every 20 μL of streptavidin magnetic beads, mix thoroughly, and rotate on a horizontal rotator at low speed for 1 min. After placing on a magnetic stand and waiting for the magnetic beads to adsorb completely, discard the supernatant.
4.4 Repeat step 4.2, washing each 20 μL of streptavidin beads twice with 40 μL of solution A, and then twice with 40 μL of solution B.
4.5 After discarding the supernatant, add 50 μL of 2× Wash Buffer to the magnetic beads (each sample) and resuspend;
5.1 Towards 3 For the RNA obtained in step 1, add 50 μL of prepared streptavidin magnetic beads to each sample;
5.2 After vortex mixing, incubate at room temperature for 20 minutes. Place the EP tube in a magnetic rack for 2-3 minutes. After the magnetic beads are adsorbed, discard the supernatant.
5.3 Add 20 μL of 1× Wash Buffer to each sample, mix well, and rotate on a horizontal rotator at low speed for 1 min. Place on a magnetic rack and wait for magnetic beads to adsorb. Discard the supernatant and repeat twice.
5.4 At this point, the nascent RNA has been captured by the streptavidin magnetic beads and can be used for downstream applications such as RT-qPCR and RNA-seq.
