| 研究課題/領域番号 |
19K16098
|
|---|
| 研究機関 | 国立研究開発法人理化学研究所 |
|---|
研究代表者 |
柴山 洋太郎 国立研究開発法人理化学研究所, 生命医科学研究センター, 研究員 (60815819)
|
|---|
| 研究期間 (年度) |
2019-04-01 – 2023-03-31
|
| キーワード | cell fixation / in-situ digestion / biotinylation of oligos / in-situ hybridization / in-situ ligation / streptavidin pull-down / RNA-seq / long noncoding RNA |
|---|
| 研究実績の概要 |
Cell fixation and in-situ digestion conditions were optimized. Various fixation reagents and duration of reactions were tested. The best condition was chosen by looking at the extent of in-situ digestion of genomic DNA following fixation. In-situ digestion was therefore optimized in combination with fixation by testing different restriction enzymes and reaction times. Genomic DNA could be digested to an average length of 1kb.
Biotinylation of pooled probes targeting long noncoding RNA was successful. This was done by first enzymatically attaching a phosphate group to the 5’ end of the pooled oligos and then chemically attaching an amino group to the phosphate group. Biotin with an ester group could be then attached to the amino groups. Biotin-labeled oligo sets were purified either by the use of streptavidin beads or by HPLC. Final yield of purified probes was enough for several downstream hybridization reactions.
For protocol development, pooled probes targeting four long noncoding RNAs with known binding sites on the human genome, were used. Both high and low copy number targets were included. Following in-situ hybridization, probes were ligated in-situ to the digested genomic DNA to which the targeted RNA bind. Successful hybridization was checked by RNA pull-down and RT-qPCR. Presence of probe-genomic DNA hybrids was also checked by RT-qPCR. Sequencing library was produced and RNA-seq conducted. Sequences, however, contained mostly background genomic sequences and very few probe-genomic DNA hybrids. This will help narrow down areas for optimization.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
RNA-seq yielded mostly genomic DNA sequences and very few probe-genomic DNA hybrids. This means there is genomic DNA contamination occurring somewhere in the protocol and needs to be optimised.
|
| 今後の研究の推進方策 |
To reduce genomic DNA contamination, more stringent pull-down using streptavidin beads will be conducted. This should lead to a better enrichment of probe-genomic DNA hybrid sequences. Once this is achieved, RNA-seq will again be conducted. Successful capturing of genomic DNA sequences associated with the four long noncoding RNA targets is a requirement before increasing target number.
|
| 次年度使用額が生じた理由 |
Due to the unexpected circumstance (of genomic DNA background occurring in the first round of RNA sequencing), sequencing was limited to one round only causing reduction in the usage of funds. In the next fiscal year, solutions to overcome this issue will be tested. If successful, more sequencing runs will be conducted.
|
