Santiago Grijalvo
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3.1 Chemical Synthesis of Lipid-siRNA 脂质-siRNA 的化学合成
3.1.1 Preparation of 3′- and 5′-Lipid RNA Oligonucleotide Conjugates 3′-和 5′-脂质 RNA 寡核苷酸共轭物的制备
Glycerol-based CPG solid supports as well as modified neutral and cationic lipid phosphoramidites were previously synthesized according to well-established experimental procedures.
基于甘油的 CPG 固体载体以及改良的中性和阳离子脂质磷酰胺酯之前已按照公认的实验程序进行合成。
Weigh 20–30 mg of commercially available CPG modified with cholesterol (Table 1; Chol) and glycerol-based CPG solid supports (Table 1; 106, 107, 110, 111, and SCR). Place them in a DNA synthesizer (see Note 12). 称量 20-30 mg 商业化的经胆固醇（表 1；Chol）和基于甘油的 CPG 固体载体（表 1；106, 107, 110, 111 和 SCR）修饰的 CPG，将它们放入 DNA 合成器中（参见 注释 12）
Dissolve the corresponding lipid phosphoramidite (Table 1; 8) in an appropriate anhydrous solvent like acetonitrile (ACN) to give a final concentration of 0.1 M (see Note 13). 将相应的脂质磷酰胺酯（表 1；8）溶解在适当的无水溶剂如乙腈（ACN）中，制备最终浓度为 0.1 M 的溶液（参见 注释 13）
The resultant lipid phosphoramidite solutions must be finally integrated into the automated synthesizer. 将所得的脂质磷酰胺酯溶液加入到自动合成仪中
Program the sequences (both guide and passenger strands) displayed on Table 1 in the DNA/RNA synthesizer. 在 DNA/RNA 合成仪中编写表 1 中显示的序列（包括导链和载链）
Remove the modified CPG solid supports (Table 1) carrying the desired oligonucleotides and the selected lipids once the automated synthesis has ended up (see Note 14).当自动合成结束后，取下携带目标寡核苷酸和脂质修饰物的 CPG 固体载体（详见 注释 14）
Transfer the solid supports to a 2 mL-screw glass vial. Add 1 mL of a mixture of concentrated ammonia (32%) in ethanol (3:1) and heat the suspension up at 55 °C for 1 h. 将固体载体转移至 2 毫升螺口玻璃瓶中。加入 1 毫升浓氨（32%）和乙醇（3:1）的混合物，并将悬浮液加热至 55°C，保持 1 小时
Filter the CPG solid supports off and wash with additional ethanol in autoclaved conical tubes. 过滤 CPG 固体载体，用额外的乙醇洗涤并收集洗液
Evaporate the mixture of solvents (water and ethanol) in a Speed Vac evaporator to dryness. 在 Speed Vac 蒸发仪中将溶液（包括水和乙醇的混合物）蒸发至干燥
Dissolve the residue containing the RNA oligonucleotide conjugates with 0.15 mL solution containing triethylamine trihydrofluoride, triethylamine, and N-methylpyrrolidone (4:3:6) for 2.5 h at 65 °C in order to remove both the silyl (TBDMS) and 3,3′-dimethoxytrytil (DMTr) protecting groups. 将含有 RNA 寡核苷酸共轭物的残余物溶解在含有三乙胺三氟化氢、三乙胺和 N-甲基吡咯烷酮（4:3:6）的 0.15 毫升溶液中，在 65°C 下反应 2.5 小时，以去除硅基（TBDMS）和二甲氧基三苯基甲基（DMTr）保护基团。
Add isopropoxytrimethylsilane (0.3 mL) to stop the deprotection reaction and diethyl ether (0.75 mL). 加入 0.3 毫升异丙氧基三甲基硅烷终止去保护反应，并加入 0.75 毫升二乙醚
Vortex the final mixture solutions and cool down overnight at 4 °C to facilitate the precipitation of the expected DMToff-RNA oligonucleotide conjugates (see Note 15). 涡旋混合溶液，并在 4°C 下冷却过夜，以促进去 DMTr-RNA 寡核苷酸共轭物的沉淀（详见 注释 15）
Centrifuge conical tubes containing RNA oligonucleotide conjugates at 14,086 × g at 4 °C for 5 min. 将含有 RNA 寡核苷酸共轭物的锥形管在 4°C 下以 14,086 × g 离心 5 分钟
Wash the corresponding pellets with diethyl ether and centrifuge again using the same conditions as described above. 用二乙醚洗涤沉淀，并再次按上述条件离心
Dry the RNA pellets carefully using a weak nitrogen stream . 使用弱氮气流仔细干燥 RNA 沉淀
3.1.2 Purification of RNA Oligonucleotide Conjugates by HPLC 通过 HPLC 纯化 RNA 寡核苷酸共轭物
Dissolve the RNA pellets obtained in Subheading 3.1.1 in RNAse-free miliQ-water or DEPC-treated water. 将在小标题 3.1.1 中获得的 RNA 沉淀溶解在无 RNA 酶 MilliQ 水或 DEPC 处理的水中
Use the appropriate desalting columns (Sephadex NAP-5 or NAP-10 columns) following the manufacturer’s instructions. 使用合适的脱盐柱（葡聚糖凝胶 NAP-5 或 NAP-10 柱），按制造商的说明进行操作
Use “Solvent A” and “Solvent B” as buffered solutions (see Subheading 2) and placed them in labeled bottles. 使用“溶剂 A”和“溶剂 B”作为缓冲溶液（详见小标题 2），并将它们放置在标记的瓶中
Analyze the corresponding RNA oligonucleotide conjugates. Run a 10-min linear gradient starting at 0% to 35% B and a flow rate of 1 mL/min (see Note 16).分析相应的 RNA 寡核苷酸共轭物。以 1 毫升/分钟的流速运行 10 分钟线性梯度，从 0%到 35%B（详见 注释 16）
Program a specific method and use a flow rate of 3 mL/min and 20-min linear gradient from 15% to 80% B maintaining 5 additional minutes to 80% B to purify the RNA oligonucleotide conjugates (see Note 16). 编写特定的纯化方法，使用 3 毫升/分钟的流速，20 分钟线性梯度从 15%到 80%B，并在 80%B 保持额外 5 分钟以纯化 RNA 寡核苷酸共轭物（详见 注释 16）
Load a syringe with 200 μL crude RNA oligonucleotide solutions (see Note 17). 使用 200 微升粗 RNA 寡核苷酸溶液装入注射器（详见 注释 17）
Collect pure RNA oligonucleotide samples in conical tubes and use a Speed Vac evaporator to concentrate the samples to dryness. 将纯化的 RNA 寡核苷酸样品收集到锥形管中，并在 Speed Vac 蒸发仪中将样品浓缩至干燥
Characterize the collected fractions by analytical HPLC and mass spectrometry (MALDI-TOF). Use a UV-spectrophotometer to calculate the final concentration of the pure RNA oligonucleotide strand. 通过分析性 HPLC 和质谱（MALDI-TOF）表征收集的纯化馏分。使用紫外分光光度计计算纯化 RNA 寡核苷酸链的最终浓度。
An example of a HPLC chromatogram and a MALDI-TOF spectrum of a modified lipid RNA passenger strand is shown in Fig. 3. 图 3 显示了改性脂质 RNA 载链的 HPLC 色谱图和 MALDI-TOF 光谱的实例
Fig. 3 (a) HPLC purification of sense-3′-lipid_C14 and OMe (110). (b) Mass spectrum (MALDI-TOF) of the main peak (expected 6993; found 7013 [M + Na+])
3.1.3 Annealing and Precipitation of siRNA Duplexes siRNA 双链的退火和沉淀
Take those lyophilized RNA strands modified with the appropriate lipid pendent groups and RNA guide strands.取用已修饰适当脂质基团并经过冻干的 RNA 链和 RNA 导链
Dissolve both strands in annealing buffer (100 μL).将两条链分别溶解在 100 微升的退火缓冲液中
Mix equimolar amounts of each RNA strands.按等摩尔比例混合两条 RNA 链
Heat the mixture of both strands up at 93 °C and cool down slowly at 25 °C for 5 h. 将混合物加热至 93 摄氏度，然后缓慢冷却至 25 摄氏度，保持 5 小时
Add 3 M AcONa solution (10 μL) and vortex the final solution. 加入 10 微升 3 M 的醋酸钠溶液，并涡旋混合溶液
Add 3 vol. of EtOH and vortex again. 加入三倍体积的乙醇，再次涡旋混合
Store the final solution at −18 °C overnight to trigger the precipitation of the lipid-siRNA conjugates.将最终溶液在−18 摄氏度过夜保存，以促使脂质-siRNA 共轭物沉淀
Isolate and dissolve the precipitated siRNA in an appropriate buffer and quantify it by UV spectroscopy at 260 nm wavelength. 分离沉淀物，将其溶解在适当的缓冲液中，并通过紫外光谱法在 260 纳米波长处定量
3.2 Evaluation of the TNF-α Inhibition Properties TNF-α抑制性能的评估
3.2.1 In Vitro Gene Silencing Studies 体外基因沉默研究
Use these general protocols with the aim to transfect the corresponding unmodified and modified lipid-siRNA conjugates in two specific adherent cell culture lines (e.g., HeLa and 4T1). Detailed protocols are described below.
本节使用了基本的实验流程，将未修饰和修饰过的脂质-siRNA 共轭物分别转染到 HeLa 和 4T1 细胞系中，以抑制 TNF-α基因表达。具体步骤如下
Use HeLa and 4T1 cell lines for in vitro gene silencing studies to inhibit TNF-α gene expression (see Note 18).选择 HeLa 和 4T1 细胞系进行 TNF-α基因表达的体外抑制实验（详见 注释 18）
Transfect a plasmid expressing such gene (pCAm TNF-α; 250 ng) using a commercially available cationic lipid, namely Lipofectamine™ 2000 when working with HeLa cell lines following the manufacturer’s instructions (see Note 18).对 HeLa 细胞系进行转染实验时，按照制造商说明，使用 Lipofectamine™ 2000（阳离子脂质转染试剂）将表达 TNF-α基因的质粒（pCAm TNF-α；250 ng）转染入细胞中（详见 注释 18）
Incubate both unmodified and siRNA bioconjugate duplexes (100 nM) displayed on Table 1 ( clbr://internal.invalid/OEBPS/html/485053_1_En_8_Chapter.xhtml#Tab1 ) with fetal calf serum (FCS) previously (see the “Pre-binding Strategy” section for further details) (Fig. 4).将表 1 中的未修饰 siRNA 双链和 siRNA 生物共轭物（100 nM）分别与胎牛血清（FCS）预孵育（详见“预孵育策略”部分的详细信息），如图 4 所示
Transfect either HeLa or 4T1 cells with the corresponding siRNA complexes.将 siRNA 复合物转染到 HeLa 或 4T1 细胞中
Use the same siRNA duplexes and transfect them using the cell lines described before without being incubated with fetal calf serum (FCS).将相同的 siRNA 双链在未与胎牛血清（FCS）孵育的情况下，进行转染实验
Transfection processes in steps 4 and 5 must be performed in the absence of Lipofectamine™ 2000. 步骤 4和步骤 5中的转染实验需在无 Lipofectamine™ 2000 的条件下进行
Fig. 4 Schematic representation of the sonication method (f-siRNA)
Pre-binding Strategy 预结合策略
Culture and grow exponentially HeLa cells under standard conditions (37 °C, 5% CO2) in DMEM as a suitable medium supplemented with 10% FBS and antibiotics (see Note 19). 在标准条件下（37°C，5% CO2），使用适当的培养基 DMEM，添加 10% FBS 和抗生素，培养并使 HeLa 细胞处于指数增长期（参见 注释 19）
Remove the growing media and use 500 μL of fresh DMEM supplemented with 10% FBS without antibiotics to perform the transfection experiments. 去除原培养基，使用 500 微升不含抗生素的新鲜 DMEM 培养基（含 10% FBS）进行转染实验
Use a 40–60% confluence to perform the transfection experiments. 当细胞融合度达到 40%至 60%时，进行转染操作
Use 250 ng of a plasmid expressing murine TNF-α (pCAm TNF-α) and mix with Lipofectamine™ 2000 in Opti-MEM (300 μL). 使用 250 纳克表达小鼠 TNF-α的质粒（pCAm TNF-α），并将其与 Lipofectamine™ 2000 在 Opti-MEM（300 微升）中混合
Incubate the resulting liposomes for 15 min at room temperature. 将混合后的脂质体在室温下孵育 15 分钟
Add 100 μL of the preformed liposome solution in three wells to get a final volume of 600 μL. 在每个孔中加入 100 微升预制的脂质体溶液，总体积为 600 微升
Incubate HeLa cells for 4 h under standard conditions (37 °C, 5% CO2) (see Note 20). 在标准培养条件下（37°C，5% CO2）孵育 HeLa 细胞 4 小时（详见 注释 20）
Wash thoroughly with 1× PBS in order to remove the lipid-based transfecting agent. 使用 1× PBS 彻底清洗细胞，以去除脂质转染试剂
Mix those siRNA duplexes (100 nM) displayed on Table 1 with FCS in the ratio of 0.66 μg siRNA/25 μL FCS. 将表 1 中的 siRNA 双链（100 nM）与胎牛血清（FCS）按 0.66 微克 siRNA/25 微升 FCS 的比例混合
Place siRNA/serum complexes on a sonication bath for 5 min (see Note 21). 将 siRNA/血清复合物在超声波浴中处理 5 分钟（详见 注释 21）
Add the resulting siRNA/FCS complexes (“f-siRNA”) to HeLa cultured cells in the presence of Opti-MEM medium and in the absence of a commercially available cationic lipid. 在无商用阳离子脂质的条件下，将生成的 siRNA/FCS 复合物（“f-siRNA”）添加到含 Opti-MEM 培养基的 HeLa 细胞中
Incubate HeLa cells containing f-siRNA for 4 h under standard conditions (37 °C, 5% CO2). 在标准培养条件下（37°C，5% CO2）孵育含f-siRNA的 HeLa 细胞 4 小时
Remove media and add fresh DMEM supplemented with 10% FBS after 4 h-incubation. 4 小时后，移除培养基，添加新鲜的 DMEM（含 10% FBS）
Incubate HeLa cells for 48 h under standard conditions (37 °C, 5% CO2). 在标准条件下（37°C，5% CO2）进一步孵育 HeLa 细胞 48 小时
Analyze the amount of TNF-α produced by HeLa cells by the enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s instructions (see Note 22). 使用酶联免疫吸附测定（ELISA）分析 HeLa 细胞中 TNF-α的表达水平，按照制造商说明操作（详见 注释 22）
Results are shown in Figs. 5 and 6.结果见图 5 和图 6
Fig. 5 Efficiency of “f-siRNA” method for silencing TNFα in 4T1 cells. Left: without sonication. Right: with sonication. 4T1 cells were transfected with 100 nM of each “f-siRNA,” after 24 h levels of TNFα were measured by ELISA. The data represent the mean ± SE, n = 3 and are compared with Scrambled sequence. p < 0.05, p* < 0.01, ***p < 0.001. ANOVA, Bonferroni post-test. w.m unmodified anti-TNFα siRNA. Chol: sense 3′-Cholesterol, 106: sense 3′lipidC14, 107: sense 3′ lipidC18, 110: sense 3′ lipidC14-OMe, 111: sense 5′ lipidC14N, Scr: Scrambled
Fig. 6 (a) Silencing of TNFα in HeLa cells. Gray (control), HeLa cells were transfected with 250 ng of TNFα plasmid pCAm using Lipofectamine™ 2000 as a vector transfection, 1 h after 100 nM of each of the siRNAs were transfected directly on the cell medium supplemented with serum. After 48 h, levels of TNFα were measured by ELISA. Black (f-siRNA), HeLa cells were transfected with 250 ng of plasmid pCAm TNFα using Lipofectamine™ 2000 as a vector, then were transfected 1 h later with 100 nM of each “f-siRNAs.” After 48 h levels of TNFα were measured by ELISA. (b) Silencing of TNFα in 4T1 cells. Gray (control), 4T1 cells were transfected with 100 nM of each siRNAs directly on the cell medium supplemented with serum. After 24 h, levels of TNFα were measured by ELISA. Black (f-siRNA), 4T1 cells were transfected with 100 nM of each “f-siRNA”, after 24 h levels of TNFα were measured by ELISA. The data represent the mean ± SE, n = 3 and are compared with Scrambled sequence *p < 0.05. Statistical analysis was performed by ANOVA, Bonferroni post-test. siRNAs: siTNF, unmodified siRNA. siTNF-Chol: sense 3′-Cholesterol, siTNF-C14: sense 3′lipid C14 (106), Scr: Scrambled
In the case of using 4T1 breast cancer cells use only steps 9–14 (without transfection of the pCAm plasmid).
在使用 4T1 乳腺癌细胞的实验中，只执行步骤 9–14（不包括 pCAm 质粒的转染步骤）
Non-pre-binding Strategy 非预孵育策略
Perform the same experiments using same siRNA duplex amounts but without sonicating them in the presence of FCS. 使用相同的 siRNA 双链量进行实验，但不在 FCS 中超声处理
Add siRNA duplexes (100 nM) to HeLa cultured cells in the absence of a commercially available cationic lipid and incubate cells and siRNA duplexes for 48 h under standard conditions (37 °C, 5% CO2). 在无商用阳离子脂质的条件下，将 siRNA 双链（100 nM）添加到 HeLa 细胞培养物中，并在标准条件下（37°C，5% CO2）孵育细胞和 siRNA 双链 48 小时
Analyze the amount of TNF-α produced by HeLa cells by the enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions . 根据制造商的说明，通过酶联免疫吸附测定（ELISA）分析 HeLa 细胞中 TNF-α的表达水平
Results are shown in Figs. 5 and 6. 结果见图 5 和图 6
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One should be extremely careful when working with RNA oligonucleotides. In this regard, glassware and plasticware should be autoclaved before putting in contact with RNA. In addition, DEPC-treated water or nuclease-free water is required to avoid the degradation of both RNA oligonucleotides and siRNA bioconjugates. To prepare DEPC-treated water, 1 mL of diethylpyrocarbonate (DEPC) is added to 1 L of water. The resultant solution is incubated at room temperature overnight and finally autoclaved. 处理 RNA 寡核苷酸时务必小心。在这方面，玻璃器皿和塑料器皿在接触 RNA 之前应先进行高压灭菌。此外，为防止 RNA 寡核苷酸和 siRNA 生物共轭物的降解，还需要使用经过 DEPC 处理的水或无核酸酶水。DEPC 处理水的制备方法是将 1 毫升二乙基焦碳酸酯（DEPC）加入 1 升水中，室温下孵育过夜，最后进行高压灭菌。
This is a solid-phase automated process in which building blocks (DNA or RNA phosphoramidites) are coupled to a growing oligonucleotide in an orderly manner leading to a specific sequence that has been previously programmed. The average yield for the coupling of RNA monomers is usually around 97–98%. Several building blocks derived from the phosphoramidite method (e.g., protected 2′-deoxynucleosides, ribonucleosides, locked nucleic acids and bridge nucleic acids) can be used in an automated oligonucleotide synthesis. 这是一个固相自动化合成过程，其中构建块（DNA 或 RNA 磷酰胺）依次与正在生长的寡核苷酸耦合，生成预先编程的特定序列。RNA 单体的耦合平均产率通常在 97-98％左右。磷酰胺法衍生的多个构建块（例如，保护的 2'-脱氧核苷、核糖核苷、锁核酸和桥核酸）可以用于自动化寡核苷酸合成。
Ancillary reagents used during the RNA synthesis are the following: in DCM (detritylation step); 0.4 M 1H-tetrazol in ACN (activation step); acetic anhydride/pyridine/tetrahydrofurane (THF) (1:1:8) (capping A); 10% N-methylimidazole in THF (capping B) and 0.01 M iodine in THF/pyridine/water (7:2:1) (oxidation step). This oxidizing agent readily oxidizes the phosphorous (III) to phosphorous (V). These ancillary reagents are available from Merck Sigma-Aldrich (Madrid, Spain), Link Technologies (Scotland, UK), Glen Research (Sterling, VI, USA), or Applied Biosystems (Foster City, CA, USA) among many others. RNA 合成过程中使用的辅助试剂包括以下：DCM（去三苯甲基步骤）；0.4 M 1H-四唑在 ACN 中（活化步骤）；醋酸酐/吡啶/四氢呋喃（THF）（1：1：8）（封闭 A）；10％N-甲基咪唑在 THF 中（封闭 B）和 0.01 M 碘在 THF/吡啶/水（7：2：1）中（氧化步骤）。这些辅助试剂可在 Merck Sigma-Aldrich（西班牙马德里）、Link Technologies（苏格兰，英国）、Glen Research（美国弗吉尼亚州斯特林）或 Applied Biosystems（美国加利福尼亚州福斯特城）等供应商处购买。
Unmodified siRNA duplexes usually have their 3′-end passenger and guide strands modified with two thymidines (dT) with the aim to protect siRNAs from degradation. However, siRNAs without bearing these dTs at the ends have been also prepared and used in cell culture experiments. 未修饰的 siRNA 双链通常在其 3'-端乘客链和引导链上修饰有两个胸苷（dT），以保护 siRNA 免受降解。然而，未修饰的 siRNA 在细胞培养实验中同样可以不具备这些 dT 端。
Common protecting groups are the following: Bz is a benzoyl group; Ac is an acetyl and dmf is a dimethylformamidine protecting groups. The 2′-OH protecting group used for natural RNA monomers is usually the tert-butyldimethylsilyl (TBDMS) protecting group although other silyl derivatives have been also reported like TOM (triisopropylsilyloxymethyl). 常见的保护基团包括以下：Bz是苯甲酰基；Ac是乙酰基，dmf是二甲基甲酰胺基。天然 RNA 单体的 2'-OH 保护基通常是tert-丁基二甲基硅基（TBDMS），尽管也有报道使用其他硅基衍生物如 TOM（三异丙基硅氧基甲基）。
Several companies can provide not only regular CPG solid supports and natural RNA phosphoramidites as described before, but also minor 2′-OMe-RNA phosphoramidites like Glen Research (Sterling, VI, USA) to afford RNA oligonucleotides with improved stabilities. 一些公司不仅提供常规的 CPG 固相载体和天然 RNA 磷酰胺，还提供一些次要的 2'-OMe-RNA 磷酰胺，如 Glen Research（美国弗吉尼亚州斯特林），以获得具有改进稳定性的 RNA 寡核苷酸。
In addition to obtaining phosphate linkages in the presence of iodine, water, and a weak base, other oxidizing reagents like tert-butyl hydroperoxide and (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO) can be also used. On the other hand, sulfurizing reagents can be integrated into automatized synthesis in order to replace one of the phosphate’s oxygens in the oligonucleotide chain by a sulfur atom and therefore obtaining RNA oligonucleotide derivatives containing phosphorothioate linkages. To do so, N,N,N,N-tetraethylthiuram disulfide (TETD), Beaucage reagent or 3-(dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) have been reported giving rise to the expected phosphorothioate linkages. It is worth mentioning that using DDTT affords better sulfurization kinetics as well as stability in solution when comparing to other sulfurizing reagents described above. 除了在碘、水和弱碱存在下形成磷酸酯键外，还可以使用其他氧化剂，如tert-丁基过氧化物和（1S）-(+)-(10-樟基磺酰)-恶唑啉（CSO）。此外，硫化试剂也可以被整合到自动化合成过程中，以将寡核苷酸链中的一个磷酸氧原子替换为硫原子，从而形成含磷硫键的 RNA 寡核苷酸衍生物。已经报道了几种硫化试剂，包括N,N,N,N-四乙基二硫化物（TETD）、Beaucage 试剂或 3-（二甲基氨基亚甲基）氨基-3H-1,2,4-二硫唑-3-硫酮（DDTT），这些试剂都能够形成预期的磷硫键。值得注意的是，与上述其他硫化试剂相比，DDTT 具有更好的硫化动力学和溶液稳定性。
Reverse and anion-exchange HPLC columns can be chosen to analyze and purify oligonucleotides (e.g., XBridge™ C18, DNAPac RP LC columns, DNASwift™ SAX-1S, Nucleosil 120-10 C18 (250 × 4 mm), among others). 可选择反相和阴离子交换 HPLC 柱来分析和纯化寡核苷酸（例如，XBridge™ C18，DNAPac RP LC 柱，DNASwift™ SAX-1S，Nucleosil 120-10 C18（250×4 mm）等）。
Polypropylene or polystyrene conical tubes should be previously autoclaved (see Note 1). 聚丙烯或聚苯乙烯锥形管应事先高压灭菌（见 注意事项 1）。
NAP DNA/RNA columns (GE Healthcare) are available in three sizes: NAP-5 (0.5 mL), NAP-10 (1.0 mL), and NAP-25 (2.5 mL). The number in brackets is the dead volume of the columns. NAP DNA/RNA 柱（GE Healthcare）有三种尺寸：NAP-5（0.5 mL）、NAP-10（1.0 mL）和 NAP-25（2.5 mL）。括号内的数字表示柱子的死体积。
Other common matrices used in MALDI-TOF mass spectrometry are: sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (ACH), 2,5-dihydroxybenzoic acid (DHB), or 3-hydroxypicolinic acid (3HPA), among others. MALDI-TOF 质谱中使用的其他常见基质包括：丁香酸（SA）、α-氰基-4-羟基肉桂酸（ACH）、2,5-二羟基苯甲酸（DHB）或 3-羟基吡啶酸（3HPA）等。
The required amount of CPG solid support for oligonucleotide synthesis will rely on DMTr-functionalization by calculating the resin loading. Interestingly, CPG solid supports modified with hydrophobic residues like cholesterol and palmitate can be found commercially available from Link Technologies (Scotland, UK) or Glen Research (Sterling, VI, USA). 用于寡核苷酸合成的 CPG 固相载体所需的量将取决于 DMTr 功能化，通过计算树脂负载。令人感兴趣的是，市场上也可以找到如胆固醇和棕榈酸等疏水性残基修饰的 CPG 固相载体，如 Link Technologies（苏格兰，英国）或 Glen Research（美国弗吉尼亚州斯特林）。
Some lipid phosphoramidites can be also dissolved in a mixture of ACN and dichloromethane (8:2) to obtain a 0.1 M concentration. In addition, many elaborated lipid phosphoramidites like 5′-tocopherol, palmitate, cholesterol, octyl-tocopherol, spermine, or stearyl can be also found commercially available from Link Technologies (Scotland, UK), Glen Research (Sterling, VI, USA), Berry & Associates (Dexter, MI, USA), ChemGenes (Wilmington, MA, USA), Metkinen (Finland), or TriLink Technologies (San Diego, CA, USA), among many others. 一些脂质磷酰胺也可以溶解在乙腈和二氯甲烷（8:2）的混合物中，以获得 0.1 M 浓度。此外，许多复杂的脂质磷酰胺，如 5'-生育酚、棕榈酸、胆固醇、辛基生育酚、精胺或硬脂酸，也可以在市场上找到，如 Link Technologies（苏格兰，英国）、Glen Research（美国弗吉尼亚州斯特林）、Berry & Associates（美国密歇根州 Dexter）、ChemGenes（美国马萨诸塞州 Wilmington）、Metkinen（芬兰）或 TriLink Technologies（美国加利福尼亚州圣地亚哥）等。
Unmodified anti-TNF-α and scrambled RNA sequences were obtained following the same experimental protocols used for lipid-RNA oligonucleotide conjugates. On the other hand, such unmodified and scrambled RNA sequences can be also obtained from commercial sources (ATD Bio, Eurogentec, Merck Sigma-Aldrich, among many others). 未修饰的抗 TNF-α和打乱序列 RNA 按照用于脂质-RNA 寡核苷酸共轭物的相同实验方案获得。另一方面，这些未修饰和打乱的 RNA 序列也可以从商业来源获得（ATD Bio，Eurogentec，Merck Sigma-Aldrich 等）。
The use of tetrabutylammonium fluoride (TBAF) instead of triethylamine trihydrofluoride does not remove the last 5′-DMTr protecting group of the RNA oligonucleotide sequence. In this sense, an additional treatment with 80% AcOH is required to remove the final DMTr protecting group. 使用四丁基氟化铵（TBAF）代替三氟乙酸三氟氢化物不会去除 RNA 寡核苷酸序列的最后一个 5'-DMTr 保护基。在这种情况下，还需要额外使用 80%醋酸以去除最终的 DMTr 保护基。
The gradient program may vary and depend on the modification chemical nature introduced in the RNA oligonucleotide. 梯度程序可能会因引入 RNA 寡核苷酸中的修饰化学性质而有所不同。
Try to avoid saturating HPLC column by getting UV-absorbances greater than 1.0. 为避免 HPLC 柱过载，应保持 UV 吸收值小于 1.0。
HeLa cells do not express naturally the murine TNF-α gene; therefore a plasmid expressing such gene (pCAm TNF-α; 250) must be previously transfected in the presence of a commercially available cationic lipid, namely Lipofectamine™ 2000. On the contrary, 4T1 murine breast cancer cells express the murine TNF-α gene and therefore there is no need for transfection with such pCAm plasmid. HeLa 细胞不天然表达小鼠 TNF-α基因，因此需要在商用阳离子脂质 Lipofectamine™ 2000 的帮助下，事先转染表达该基因的质粒（pCAm TNF-α；250）。相比之下，4T1 小鼠乳腺癌细胞天然表达小鼠 TNF-α基因，因此不需要转染该 pCAm 质粒。
A penicillin streptomycin solution (between 0.5 and 1.0 mL) is added to cell culture media (100 mL) to prevent bacterial contamination. A final concentration of penicillin (50–100 IU/mL) and streptomycin (50–100 μg/mL) must be obtained. 为防止细菌污染，应在 100 mL 细胞培养基中加入 0.5 至 1.0 mL 的青霉素链霉素溶液，使青霉素达到 50-100 IU/mL，链霉素达到 50-100 μg/mL 的最终浓度。
A 1-h incubation involving murine expressing TNF-α plasmid and Lipofectamine™ 2000 has been also carried out obtaining similar transfection efficiencies. 还进行了小鼠 TNF-α表达质粒与 Lipofectamine™ 2000 共同孵育 1 小时的实验，获得了类似的转染效率。
Lipid-siRNA duplexes were stable during the sonication process as observed by gel electrophoresis after phenol extraction of the serum proteins. 脂质-siRNA 双链在超声处理过程中保持稳定，这通过蛋白质酚提取后凝胶电泳实验观察到。
The results from these experiments show that the amount of TNF-α produced by cells after 48 h with lipid-siRNA conjugates combined with 10% FCS differ from those transfection processes performed in the absence of the pre-binding strategy. In this sense, a 40–50% inhibition of the TNF-α production compared with the scrambled control was observed only if siRNAs were transfected using the pre-binding approach. Interestingly, the nature of lipid pendent groups at the 3′- and 5′-termini of the passenger strand is also important in the inhibition of the TNF-α expression. The strongest inhibition (80% in 4T1 cells) was observed when a siRNA duplex was modified with the hydrophobic lipid chain C14 at the 3′-termini of the sense strand (Figs. 5 and 6).这些实验结果表明，使用脂质-siRNA 共轭物与 10% FCS 孵育 48 小时后，细胞产生的 TNF-α量与未采用预结合策略的转染过程相比有所不同。在这种情况下，只有使用预结合方法转染 siRNA 时，TNF-α产生量相比打乱对照降低了 40-50％。有趣的是，乘客链 3'-和 5'-末端的脂质链性质在抑制 TNF-α表达中也起着重要作用。当 siRNA 双链在反义链的 3'-末端修饰了疏水性脂质链 C14 时，在 4T1 细胞中观察到最强的抑制作用（80％）（图 5 和图 6）。
