Difference between revisions of "Team:NJU-China/Collaborations"

In order to design short hairpin RNA (shRNA) conform to demands in our experiments, we cooperated with SYSU-Software from Sun Yat-sen University to develop a corresponding software. We provided algorithms and design requirements, and then SYSU-Software wrote this delicate software for us. With their help, we successfully designed K-ras shRNA, whose sequence and predicted structure are shown below (Fig. 1). We validated that this shRNA was able to down-regulate the expression level of K-ras in A549 effectively. We thank SYSU-Software for receiving us with attention and for such strong contribution to our work.

Since team NUDT_CHINA met some troubles in assessing the efficiency of the single guide RNAs designed for their experiments, we performed a Cas9-digest assay to assist them validating the effectiveness of their sgRNAs. Also, we helped them to optimize their own protocol for in vitro expression of sgRNAs, so that their project could be performed more smoothly.
With the expressing plasmids provided by team NUDT_CHINA, in vitro transcription based on T7 RNA polymerase was conducted to obtain enough amount of sgRNAs. Two standard sgRNAs, with the binding efficiency of 20% and 70% were also transcribed to evaluate the efficiency of sgRNAs. Once transcribed, purified Cas9 protein was used to digest target DNA (provided by NUDT_CHINA team) with the guidance of sgRNAs.
Results showed that when transcribed correctly (Fig. 2A), sgR-1 and sgR-4 could successfully mediate the digestion of target DNA by Cas9 protein, while the negative control adding no sgRNA showing no digestion output. The binding efficiency was estimated to be between 20% and 70%, while the binding efficiency of sgR-1 is significantly higher than sgR-4 (Fig. 2B). Those results indicated that sgR-1 could be a better choice to be used in their project.

(A) Agarose electrophoresis showing the result of in vitro transcription of sgRNAs. “Stand #1” and “Stand #2” stood for two standard sgRNAs with the binding efficiency of 20% and 70% that were transcribed to evaluate the efficiency of sgRNAs in down-stream experiments.
(B) Agarose electrophoresis showing the result of Cas9 digestion mediated by previously transcribed sgRNAs. N.C. stood for the negative control adding no sgRNA.

In addition, we'd like to express our sincere thanks to NUDT_CHINA for their generous help in Nanosight, enabling us to have a much more precise determination of the quantity and size of exosomes we collected. You can see the detailed information in our "Results" page.

We much appreciate that SCUT-China from South China University of Technology provided vigorous support and supplement in our modeling. You can see the detailed information in our "Modeling" page.

Since JSNU-China from Jiangsu Normal University confronted some troubles in their research on neuroblastoma cells and we had done some serious experiments in this field, we offered them cells and gave them some pieces of advice.