本研究由 R-loop 搜尋程式所預測的 53 個基因中，挑選 13 個基因做為研究 目標，目前尚未發現 GMSA 實驗結果與候選基因之間的關聯性。為了進一步驗 證 R-loop 搜尋程式是否能更準確預測可能形成的 R-loop 的基因，可以從 53 個 基因中挑選更多基因進行研究，以增加樣本數。另外也從非預測基因中，選擇更 多基因進行研究，觀察是否有不具有原先設定的序列特徵而能形成 R-loop 的基 因，如 COOLAIR (Sun et al., 2013)。以 COOLAIR 進行 GMSA 結果顯示，沒有色 帶模糊出現 (Appendix )，暗示著會形成 R-loop 的基因不一定有序列特徵性。
除了 GMSA 外，還可以利用 native bisulfite sequencing assay 尋找細胞內的 R-loop 。當細胞內形成 R-loop 時，非模板股會形成單股 DNA 裸露在外，而此 時以 bisulfite 處理後，會使 DNA 上的 dC 轉換成 dU，接著進行進行聚合酶連鎖 反應，得到的產物則會出現 dC 到 dT 轉換的片段 (簡稱 C-T 轉換)。我們可以藉 由這些轉換片段知道由程式預測出的基因是否如預期一樣有 R-loop 的形成。
Native bisulfite sequencing assay 的第一步是抽取阿拉伯芥的基因組 DNA (genomic DNA) ，謝閔翔 (2015)的實驗流程是將植株均質化後，加入 urea-containing extraction buffer (7 M urea, 0.3 M NaCl, 50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 及 1% sarcosine)，接著使用 Phenol:Chloroform:Isoamyl Alcohol(PCI)去除 蛋白質跟脂質，異丙醇沉澱後即可得到基因組 DNA。使用 urea extraction buffer 是藉由 urea 讓蛋白質後，以及破壞 DNA 與水分子之間的氫鍵，再藉由 PCI 萃取 蛋白質，進而純化出基因組 DNA。
但有另一種更適合的抽取阿拉伯芥的基因組 DNA 的實驗方法: 使用 Honda
buffer (0.44 M Sucrose, 1.25 % Ficoll, 2.5 % Dextran T40, 20 mM Hepes KOH pH7.4, 10 mM MgCl2, 0.5 % Triton X-100, 5 mM DTT 及 Protease Inhibitors) 分離細胞 核，並以 miracloth 去除雜質，再使用 SDS 打破細胞核，以及利用 proteinase K 分 解蛋白質。最後使用 phenol/chloroform 萃取去除雜質，進行酒精沉澱即可得到基 因組 DNA。
本研究曾沿 續謝閔翔選擇的 LBD18 為研究目標，進行 native bisulfite sequencing assay，希望可以利用更適合植物的實驗方法，找出 LBD18 在細胞內 會形成 R-loop 的證據，但未能成功。原先認為是 native bisulfite sequencing assay 實驗上的條件不適當，但以含有 LBD18 序列的質體進行 GMSA 後觀察不到色帶 模糊出現，有可能是 LBD18 本來就為不會形成 R-loop 的基因，未來可以挑選 RL-13 或 DOT1 作為研究目標進行 native bisulfite sequencing assay。
另外，未來也希望可以進行 DNA/RNA immunoprecipitation (免疫沉澱)，
DNA/RNA immunoprecipitation 的實驗原理為利用可以偵測 DNA/RNA 雜合結構 的抗體, S 9.6, 結合 DNA/RNA 雜合體，搭配針對目標基因設計的引子對，進一 步使用即時定量聚合酶鏈鎖 (real time qPCR)，觀察實驗結果推測目標基因是否 會形成 R-loop。另外，免疫沉澱完所得片段，可利用次世代定序 (Next Generation Sequencing , NGS)，尋找出阿拉伯芥中可能會形成 R-loop 的基因。
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Figures
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Figure 1. (A) Formation of DNA/RNA hybrid in RL-1 observed by gel mobility shift assay protocol I. (B) Formation of DNA/RNA hybrid in RL-1 observed by gel mobility shift assay protocol II. (C) Formation of DNA/RNA hybrid in RL-1 observed by gel mobility shift assay protocol III. (D) A diagram showing the primer set flanking the DNA fragment containing the predicted RIZ+REZ zone in RL-1. (E) The diagram showing the cloned RL-1 fragment in relation to the T7 and SP6 promoter site.
Detection of the presence of DNA/RNA hybrid with pTA-RL-1 plasmid by GMSA.
The purified plasmid was subjected to in vitro transcription either by T7 RNA polymerase or SP6 RNA polymerase. The reaction mixture was then treated with RNase A or RNase A/RNase H. The supercoiled form of DNA is indicated by an arrow. The open circular form of DNA is indicated by a dot arrow.
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Figure 2. (A) Formation of DNA/RNA hybrid in RL-2 observed by gel mobility shift assay. (B) Formation of DNA/RNA hybrid in RL-2 observed by gel mobility shift assay protocol II. (C) Formation of DNA/RNA hybrid in RL-2 observed by gel mobility shift assay protocol III. (D) A diagram showing the primer set flanking the DNA fragment containing the predicted RIZ+REZ zone in RL-2. (E) The diagram showing the cloned RL-2 fragment in relation to the T7 and SP6 promoter site.
Detection of the presence of DNA/RNA hybrid with pTA-RL-2 plasmid by GMSA.
The purified plasmid was subjected to in vitro transcription either by T7 RNA polymerase or SP6 RNA polymerase. The reaction mixture was then treated with RNase A or RNase A/RNase H. The supercoiled form of DNA is indicated by an arrow. The open circular form of DNA is indicated by a dot arrow.
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Figure 3. (A) Formation of DNA/RNA hybrid in RL-3 observed by gel mobility shift assay. (B) Formation of DNA/RNA hybrid in RL-3 observed by gel mobility shift assay protocol II. (C) Formation of DNA/RNA hybrid in RL-3 observed by gel mobility shift assay protocol III. (D) A diagram showing the primer set flanking the DNA fragment containing the predicted RIZ+REZ zone in RL-3 (E) The diagram showing the cloned RL-3 fragment in relation to the T7 and SP6 promoter site.
Detection of the presence of DNA/RNA hybrid with pTA-RL-3 plasmid by GMSA.
The purified plasmid was subjected to in vitro transcription either by T7 RNA polymerase or SP6 RNA polymerase. The reaction mixture was then treated with RNase A or RNase A/RNase H. The supercoiled form of DNA is indicated by an arrow. The open circular form of DNA is indicated by a dot arrow.
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Figure 4. (A) Formation of DNA/RNA hybrid in RL-4 observed by gel mobility shift assay. (B) Formation of DNA/RNA hybrid in RL-4 observed by gel mobility shift assay protocol II. (C) Formation of DNA/RNA hybrid in RL-4 observed by gel mobility shift assay protocol III. (D) A diagram showing the primer set flanking the DNA fragment containing the predicted RIZ+REZ zone in RL-4. (E) The diagram showing the cloned RL-4 fragment in relation to the T7 and SP6 promoter site.
Detection of the presence of DNA/RNA hybrid with pTA-RL-4 plasmid by GMSA.
The purified plasmid was subjected to in vitro transcription either by T7 RNA polymerase or SP6 RNA polymerase. The reaction mixture was then treated with RNase A or RNase A/RNase H. The supercoiled form of DNA is indicated by an arrow. The open circular form of DNA is indicated by a dot arrow.
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Figure 5. (A) Formation of DNA/RNA hybrid in RL-6 observed by gel mobility shift assay. (B) Formation of DNA/RNA hybrid in RL-6 observed by gel mobility shift assay protocol II. (C) Formation of DNA/RNA hybrid in RL-6 observed by gel mobility shift assay protocol III. (D) A diagram showing the primer set flanking the DNA fragment containing the predicted RIZ+REZ zone in RL-6. (E) The diagram showing the cloned RL-6 fragment in relation to the T7 and SP6 promoter site.
Detection of the presence of DNA/RNA hybrid with pTA-RL-6 plasmid by GMSA.
The purified plasmid was subjected to in vitro transcription either by T7 RNA polymerase or SP6 RNA polymerase. The reaction mixture was then treated with RNase A or RNase A/RNase H. The supercoiled form of DNA is indicated by an arrow. The open circular form of DNA is indicated by a dot arrow.
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Figure 6. (A) Formation of DNA/RNA hybrid in RL-7 observed by gel mobility shift assay. (B) Formation of DNA/RNA hybrid in RL-7 observed by gel mobility shift assay protocol II. (C) Formation of DNA/RNA hybrid in RL-7 observed by gel mobility shift assay protocol III. (D) A diagram showing the primer set flanking the DNA fragment containing the predicted RIZ+REZ zone in RL-7. (E) The diagram showing the cloned RL-7 fragment in relation to the T7 and SP6 promoter site.
Detection of the presence of DNA/RNA hybrid with pTA-RL-7 plasmid by GMSA.
The purified plasmid was subjected to in vitro transcription either by T7 RNA polymerase or SP6 RNA polymerase. The reaction mixture was then treated with RNase A or RNase A/RNase H. The supercoiled form of DNA is indicated by an arrow. The open circular form of DNA is indicated by a dot arrow.
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Figure 7. (A) Formation of DNA/RNA hybrid in RL-13 observed by gel mobility shift assay. (B) Formation of DNA/RNA hybrid in RL-13 observed by gel mobility shift assay protocol II. (C) Formation of DNA/RNA hybrid in RL-13 observed by gel mobility shift assay protocol III. (D) A diagram showing the primer set flanking the DNA fragment containing the predicted RIZ+REZ zone in RL-13. (E) The diagram showing the cloned RL-13 fragment in relation to the T7 and SP6 promoter site.
Detection of the presence of DNA/RNA hybrid with pTA-RL-13 plasmid by GMSA. The purified plasmid was subjected to in vitro transcription either by T7 RNA polymerase or SP6 RNA polymerase. The reaction mixture was then treated with RNase A or RNase A/RNase H. The supercoiled form of DNA is indicated by an arrow. The open circular form of DNA is indicated by a dot arrow.
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Figure 8. (A) Formation of DNA/RNA hybrid in RL-14 observed by gel mobility shift assay. (B) Formation of DNA/RNA hybrid in RL-14 observed by gel mobility shift assay protocol II. (C) Formation of DNA/RNA hybrid in RL-14 observed by gel mobility shift assay protocol III. (D) A diagram showing the primer set flanking
Figure 8. (A) Formation of DNA/RNA hybrid in RL-14 observed by gel mobility shift assay. (B) Formation of DNA/RNA hybrid in RL-14 observed by gel mobility shift assay protocol II. (C) Formation of DNA/RNA hybrid in RL-14 observed by gel mobility shift assay protocol III. (D) A diagram showing the primer set flanking