Team:SCUT-China B/Protocol
1. transfection efficiency exploration
We explored the best ratio of GenjetTM transfection reagent to DNA. According to the manufactures’s protocol, the ratio varies from 2:1 to 3:1, therefore we set a series of gradients, 2.4:1, 2.6:1, 2.8:1, 3:1, and we transfected pEGFP-N2 into A549. After 24h, we detected the flurescence, and found that the best ratio for A549 cell is 2.6:1.
(1)Transfection reagent: DNA= 3:1. The pictures were taken after 48h.
(2)Transfection reagent: DNA= 2.8:1. The pictures were taken after 48h.
(3)Transfection reagent: DNA= 2.6:1. The pictures were taken after 48h.
(4)Transfection reagent: DNA= 2.4:1. The pictures were taken after 48h.
2. RT-qPCR
Total RNA was extracted using MiniBEST Universal RNA Extraction Kit (TAKARA) First-strand cDNA was synthesized using a PrimeScriptTM RT Reagent Kit (TAKARA). Synthesized cDNA was subjected to RT-PCR using Premix Ex Taq™ Version 2.0 (TAKARA) with specific primers.
The primer sequences used for the amplification of Bcl-2, Bax, and GAPDH were as follows: Bcl-2 forward, 5'-TTCTCTCGTCGCTACCGTCGC-3' and reverse, 5'-CCTCCCCCAGTTCACCCCATC-3'; Bax forward, 5'-CTTTTTGCTACAGGGTTTCA-3' and reverse, 5'-CCATGTTGTTGTCCAGTTCAT-3'; GADPH forward, 5'-CGACCACTTTGTCAAGCTCA-3' and reverse, 5'-AGGGGTCTACATGGCAACTG-3'.
Moreover, qPCR was performed with SYBR Premix Ex Taq™ (TAKARA) on Roche Lightcycler 96 according to the manufacturer’s protocol.
3. western blotting
Cells (1.2x106) were harvested and lysed in lysis buffer(1%PMSF)on ice for 20 min, followed by a centrifugation at 14,000g for 10 min at 4 °C and the supernatants were then collected. The protein concentration in the supernatant was determined with the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). The samples (40 μg protein for each sample) were then subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE 12%) and transferred to nitrocellulose membranes (Bio-Rad, Richmond, CA, USA) for 50min in 150mA , blocked with 5% (w/v) nonfat milk in Tris-buffered saline (TBST containing 0.2% Tween 20) at room temperature for 110min and then washed withTBST buffer(12min X 3）. The membrane was incubated with antibodies against Bax(ab32503 abcom 1:2000),Bcl-2(ab32124 abcom 1:2000) ,caspase3(ab13847 abcom ) at room temperature for 100min. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies(ab6721 abcom 1:3000) for 90min. Expression of β-actin( ab8227 abcom 1:2000) was used as a loading control. After the incubation, the signals were visualized by using enhanced chemiluminescence (ECL) detection kit (Pierce,Rockford,IL,USA ).
4. Fluorescence analysis by image pro plus
We use Image Pro Plus to analyze the fluorescence data of the tumor specific CRISPRa.
1. Convert the picture into Gray scale 8
2. Invert contrast
3. Intensity calibration
4. Select measurement
5. Count
6. View Statistics—IOD
5. Apoptosis detection
(Pretreatment)
1. Harvest the cells
2. Use the PBS to wash the cells
3. Then use pure 1640 culture medium to resuspend(cells stayed in this enviroment before sent to company to detect)
(Treatment)
4. Centrifuge samples at 1000×g for 5 minutes and decant the supematant
5. Add PBS to count the number of the cells
6. Extract 5-10 ten thousand of cells and centrifuge samples at 1000g for 5 minutes and decant the supermatant.
7. Gently resuspend the cells in 100μl 1X Annexin V buffer
8. Add 5μl Annexin V-FITC and 5μl PI staining,gently mix them up
9. Incubate tubes in the dark for 15minutes at room temperature
10. Use the flow cytometry to detect immediately after the cubation.The Annexin-V-FITC let out green lights while the PI staining let out red staining
