Depending on our design, we standardize the whole experiment, and make it into a kit. During the designing, we communicated with the experienced professors and revise operation procedure according to the principle.
Instruction on CiC Kits
CiC Kits contains 3 key components: i) the ssDNA aptamers for specifically targeting the receptors of CTCs and visualizing them; ii) once successfully targeting CTCs, the ssDNA aptamers will expose the sticky end further for triggering the adhesion of sticky-end pairing ssDNA; iii) the pairing ssDNA can induce rolling circle amplification, subsequent multi-primed chain amplification, making the formation of CiC around CTCs. Finally, CiC can enlarge CTC size for centrifugal isolation and meanwhile keep their bioactivity. In this work, we achieve a feasible and economical CiC for clinical CTCs-capture and analysis.
Steps:
- Take 1 ml of fresh anticoagulant, add 3-5 ml of red blood cell lysate and mix.
- Centrifuge the mixture at 800-1000 rpm for 3-5 minutes and discard the upper red liquid.
- Add 1 ml of red blood cell lysate into the solution and gently blow.
- Centrifuge the solution at 800-1000 rpm for 3-5 minutes, and discard the supernatant.
- If the precipitate remains red, repeat step 3 and 4.
- Use Hank's solution to wash for 2-3 times, and discard the upper red liquid.
- Resuspend the cells with 100 μL of binding buffer and incubate them at 37 ° C for 30 min.
- Centrifuge the solution at 800 rpm for 3 min, resuspend the cells with the aptamer solution and incubate it for 2 h at 37 °C.
- Centrifuge the solution at 800 rpm for 3 min, add 130 μL of TE buffer solution and 10 μL of circular template into it and incubate it for 2 h at 37 °C.
- Add 1 μL of phi 29 DNA polymerase, 20 μL of phi 29 buffer (10X), 20 μL of dNTP and 2 μL of BSA into the solution, incubate it for 1 h at 37 °C.
- Add 5 μL of primer 2, 5 μL of primer 3 into the solution, incubate it for 5 h at 37 °C.
- Centrifugation the solution at 12.500 rpm for 3 min, the precipitate is hydrogel-encapsulated CTCs.
- Add 5 μL of Exonuclease I, 2 μL of Exonuclease III into the solution to remove the DNA hydrogel from the CTC.
- Centrifuge the solution at 800 rpm for 3 min and obtain living CTCs.