STAR-Click Nascent Transcript Capture Kit

Note:

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. Please keep the reagent on ice during use.

This product is for research use only.

Kit 1: Store at 2~8℃, valid for 12 months;

Kit 2: Store at -25~-15℃, valid for 12 months.

Experimental process: Steps:

1. Culture and RNA extraction

1. EU incubation (different conditions for different samples)

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 common cell EU is 1 mM and incubate for 2 hours. Or according to literature reports, the final concentration of EU and incubation time can also be adjusted. For the first experiment, a preliminary experiment can be performed according to the recommended EU dose and time; for example, after the cells in the T25 flask have their culture medium removed, the prepared culture medium containing 1 mM EU is added to the culture flask and incubated for 2 hours. ^* Follow step 2 for subsequent experiments.

The following table shows the recommended EU doses and times for testing:

* 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-cancer cells include human prostate cancer cells (451, SW756, ME-180), 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), neural cells (U87, NSC-13, U251, PC12), etc.

1.2 Animal tissue EU incubation

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 then 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, it is recommended to explore the concentration of EU for the first time, or directly use a concentration of 200 mg/kg for testing. After 4 hours, ^* Or after an appropriate time determined by the specific experiment, quickly kill the mouse and remove the desired tissue. Follow step 2 in 1 for subsequent experiments.

*Experiments have been verified on 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.

*Experiments have verified the hydroponic and soil-cultured ginseng, various flowers and food crops, etc.

2 RNA extraction

2.1 For cultured cell samples, remove the culture medium, lyse the cells with 1 ml TRIZOL, place in an enzyme-free EP tube, and let stand at room temperature for 5 min; for plant and animal samples, after obtaining the corresponding tissue, wash the tissue 2-3 times with enzyme-free PBS, place it in 1 mL TRIZOL and grind it with a grinding instrument until no tissue blocks are visible, place it in an enzyme-free EP tube, and let stand at room temperature for 5 min;

2.2 Add 200 μL chloroform, vortex to mix, and let stand at room temperature for 3 min;

2.3 Centrifuge at 14000g for 5 min, take the supernatant, add an equal volume of isopropanol, and vortex to mix;

2.4 Place on ice for 10 min, centrifuge at 14000g for 20 min, and discard the supernatant;

2.5 Wash the precipitate with 75% ethanol and centrifuge at 14000g for 5 min;

2.6 Repeat step 6;

2.7 Discard the supernatant, wait for the ethanol to evaporate, and add 50 μL of DEPC water to dissolve the RNA.

2.8 rRNA removal: It is recommended to use the Qihengxing Ribosomal RNA Removal Kit to remove rRNA (this step is a recommended option, rRNA removal can improve the detection sensitivity of the target RNA and reduce the amount of sequencing).

3 Click React

3.1 Click reaction solution preparation

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 TRIZOL to the click reaction solution and vortex to mix;

3.5 Repeat the RNA extraction process (TRIZOL extraction).

4. Prepare Streptavidin Magnetic Beads (SMB)

4.1 Vortex to fully suspend the streptavidin magnetic beads. Do not centrifuge to prevent the magnetic beads from settling.

4.2 Take 20 μL of resuspended magnetic beads from each sample and place them in a new EP tube. For the cleaning of SMB magnetic beads required for multiple samples, they can be done together, such as dispensing the magnetic beads of 6 samples (120 μL) or 12 samples (240 μL) into one tube for cleaning. Add 20 μL of 2×Wash Buffer to each 20 μL of streptavidin magnetic beads, mix well, place on a horizontal rotator and rotate at low speed for 1 min, then place on a magnetic stand and wait for the magnetic beads to be adsorbed, and discard the supernatant;

4.3 After removing the EP tube containing magnetic beads, add 40 μL of 2×Wash Buffer to every 20 μL of streptavidin magnetic beads, mix well, and place on a horizontal rotator for low speed rotation for 1 min. After placing on a magnetic stand and completing the magnetic beads adsorption, discard the supernatant;

4.4 Perform the same operation as step 4.2, but wash each 20 μL of streptavidin beads twice with 40 μL of solution A, and then wash 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) to resuspend;

5. Nascent RNA Capture

5.1 Towards 3 RNA obtained in step 1 was added to each sample with 50 μL of the prepared streptavidin magnetic beads;

5.2 After vortex mixing, incubate at room temperature for 20 min, place the EP tube in the magnetic rack for 2-3 min, and discard the supernatant after the magnetic beads are adsorbed;

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 the magnetic beads to be adsorbed. Discard the supernatant. Repeat twice.

5.4 At this point, the nascent RNA has been captured by the streptavidin magnetic beads. The captured nascent RNA can be used for downstream applications such as RT-qPCR, RNA-seq, etc.