Single-cell mRNA differential display assay
Several methods exist to show changes in the expression levels of unknown genes in tissue samples in response to physiological stimuli. However, the morphological and functional heterogeneity of cells within many tissues makes it often necessary to examine differences in gene expression at the individual cell level. We now introduce a new method that is routinely used to examine differential expression of unknown genes at the single-cell level.
Modern Neuroscience Research Techniques
Author(s): U. Windhorst & H. Johansson Translated by Z. Q. Zhao Jun Chen
Operation method
Single-cell mRNA differential display assay
Principle
Flowchart
Materials and Instruments
Solutions and Buffers Move I. Isolation of RNA Control and experimental cells were collected into 1.5 ml microcentrifuge tubes containing 45ul2 mmol/L DTT. The cells were thermolysed in a water bath at 95℃ for 2 min. Add the following to each tube: Incubate at 37°C for 30 min. Add the following substances: Incubate at 37°C for 15 min. Add DEPC-treated H2O to a final volume of 200ul. Add 200ul of phenol/chloroform/isoamyl alcohol (25:24:1). Shake for 30s. Microcentrifuge 14OOOOg for 5 min. Transfer the supernatant to a clean 1.5 ml tube and add 200ul of chloroform/isoamyl alcohol (24:1). Shake for 30s. Centrifuge in a microcentrifuge at 14,000 tons for 2 min. Add 1ul(10ug) of glycogen, 20ul of 3mol/L sodium acetate (pH5.2) and 600ul of 100% ethanol to precipitate RNA at -20℃ overnight. Note: Glycogen addition and overnight stay are not required in the following precipitation steps. Centrifuge at 14000 gX30 min in a microcentrifuge at 4°C. Carefully remove the supernatant and add 200ul of 70% ethanol. Centrifuge briefly in a microcentrifuge. Carefully remove the supernatant and dry the precipitate for 5 min at room temperature. Redissolve the precipitate as required in the next step. 1. Add 10ul of DEPC-treated water to re-solubilize the RNA precipitate, and add 1ul (10ug) of T7digo (dT)18. 2. Denature the RNA in a water bath at 95℃ for 2 min and cool rapidly on ice. 3. Add the following substances: 4. incubate at 37°C for lh. 5. Add the following substances: 6.16°C water bath for 2 h. 7. Perform phenol/chloroform/isoamyl alcohol extraction, chloroform/isoamyl alcohol extraction, and ethanol precipitation according to steps 7-19 of RNA isolation above. 8. Add 10ul of water to re-dissolve the double-stranded cDNA precipitate and dialyze it with 50 ml of TE liquid (without RNAase) for 4 h. (Gently transfer the cDNA onto a 0.025um microporous filter, which is floated in a conical tube containing 50 ml of TE liquid without RNAase.) After 4 h, transfer the sample into a 1.5 ml reaction tube, rinse the filter with 5 ml of DEPC-treated water and add the washings to the same tube. Rinse the filter with 5 DEPC-treated water and add the wash solution to the same tube). 1. Add the following to every 15.2ul of double-stranded cDNA solution obtained in the above steps: 2. Incubate at 37℃ for 4 h. 3. Add the following substances: 4. Incubate at 37°C for 30 min. 5. Perform phenol/chloroform/isoamyl alcohol extraction, chloroform/isoamyl alcohol extraction, and ethanol precipitation according to steps 7-19 of RNA isolation above. 1. Add 10ul of DEPC-treated water to re-solubilize the aRNA precipitate, and then add 1ul (10ng) of hexanucleic acid primer. 2. Denature the aRNA in a water bath at 95°C for 2 min and cool rapidly on ice. 3. Add the following substances: 4. incubate at 37°C for lh. 5. Perform phenol/chloroform/isoamyl alcohol extraction, chloroform/isoamyl alcohol extraction and ethanol precipitation according to steps 7-19 of the RNA isolation above. 6. Perform dialysis as described in step 8 of cDNA synthesis. 7. Transfer the cDNA to a clean reaction tube and add DEPC-treated water to a final volume of 50 ul. 1. Add the following substances to a 0.5 ml reaction tube: 2. When the thermal cycler used does not have a preheated top, a drop of vegetable oil may be added to the reaction tube to prevent evaporation of water. 3. Place the reaction tube into a thermal cycler preheated to 94°C and incubate for 3 min before performing PCR. 4. Cycle 40 times. 5. Add 8pd formamide spiking buffer, 94°C, 5 min to denature DNA. 6.5% denatured polyacrylamide sequencing gel electrophoresis, 6ul of sample per well to walk the gel until xylene cyanine FF reaches 3/4 of the gel length. 7. Cover the wet gel with a plastic sheet without fixation, radiographically autoradiograph overnight, and label the corners of the gel with fluorescent ink. 8. Use the highlights as markers to accurately arrange the exposed film and gel, cut off the target strip and put it into a clean 1.5 ml reaction tube. 9. Add 500 gel elution buffer to each strip, and leave it in 95℃ water bath for 10 min, and at room temperature for 16 hours. 10. Add 200 ul of gel elution buffer containing the target PCR product to a clean 1.5 ml reaction tube, add 1 ul (10 ul) of glycogen, and precipitate the DNA as described in steps 7 to 19 above for RNA isolation. 11. The DNA precipitate is reconstituted in 10ul of DEPC-treated water. The PCR product can be stored at -20°C at this point, and a small portion of the sample (e.g., 1-3ul) can be used for re-amplification (with the same primers as in the DD-PCR reaction) and subsequent cloning or sequencing. The number of PCR cycles for re-amplification is strictly based on the recovery of the initial PCR product from the polyacrylamide gel. Generally, 20 to 40 cycles are sufficient to obtain sufficient product for subsequent cloning. We recommend the use of commercially available kits for direct cloning of re-amplified PCR products, such as the TOPOTA Cloning Kit from Invitrogen. We have used the single-cell mRNA differential display technique to identify genes involved in the process of synapse formation between two known neurons in mollusks (VanKesterenetaL1996). After target cells were selected, it was found that the formation of synaptic connections between neurons depended on altered gene expression in both presynaptic as well as postsynaptic neurons. Knowing the nature of these changes is important for our understanding of neural development, neuronal plasticity, and regenerative IP in the nervous system. Because the methods described above include aRNA amplification, this greatly increases the reproducibility of the form of differential display. We have identified more than 30 up- and down-regulated genes (Figs. 3-5). These genes are currently being characterized. Common Problems List of reagents For more product details, please visit Aladdin Scientific website.
Water Baths PCR Thermal Cyclers Microcentrifuges Plastics and Filters
Results
![溶液和缓冲液 — 5 X DNA 酶 1/蛋白酶 K 缓冲液： 250 mmol/L Tris-HCl (pH 7.5)， 10 mmol/L CaCl2, 100 mmol/L MgCl2 _ 5 X 第一链缓冲液： 250 mmol/L Tris-HCl (pH 8.3), 375 mmol/L KC1 , 15 mmol/L Mg〇2 一 5 X 第 二 链 缓 冲 液 ： 94 mmol/L Tris-HCl (pH 6.9)， 453 mmd/L KC1 , 23 mmol/L MgCL, 750 mmol/L /3-NAD, 50 mmol/L (NH4)2S〇 4 — 5 X T 7 RNA聚合酶缓冲液： 200 mmol/L Tris-HCl (pH 8.0), 40 mmol/L MgCl2, 10 mmol/L 亚精胺- (HCl)3， 125 mmol/L NaCl —10 XPCR 缓冲液： 200 mmol/L Tris-HCl (pH 8.4)， 500 mmol/L KC1， 20 mmol/L MgCl2 - TE: 10 mmol/L Tris-HCl (pH 7.5), I mmol/L EDTA 一凝胶冼脱缓冲液： 10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, I mmol/L EDTA —200 mmol/L EDTA/5% SDS 一 经DEPC处理过的重蒸（ 馏）水 —100 mmol/L DTT —2 mmol/L DTT —3mol/L 乙 酸 钠 （ pH5.2) —350 mmol/L KCl —TE饱和酚 —氯仿 —异戊醇 一 肝 糖 原 （10 mg/ml) 一100 % 乙醇 一 70 % 乙醇 一10 mmol/L dNTPs (10 mmol/L dATP, 10 mmol/L dCTP, 10 mmol/L dGTP, 10 mmol/L dTTP) —200 /_ onol/L dNTPs (200 fxmol/L dATP, 200 卩 mol/L dCTP, 200 fimol/L dGTP, 200 /^mol/L dTTP) —10 mmol/L NTPs (10 mmol/L ATP, 10 mmol/L CTP, lOmmol/L GTP, 10 mmol/L UTP) — U -32P] dCTP (3000 Ci/mmol; 10 mCi/ml) ICi = 3.7 X IO10Bq —甲酰胺加样缓冲液（9 5 % 甲酰胺； ZOmmol/LEDTA; 0.05%二甲苯苯胺FF) 4 0 % 聚丙烯酰胺溶液（3 8 % 单丙烯酰胺， 2 % 双丙烯酰胺） —iV, JV, N'， AT-四亚甲基二胺（ TEMED) —10% 过硫酸铵 一荧光墨水（ fluorescent ink](http://img.dxycdn.com/trademd/upload/userfiles/image/2016/07/A1468375059786qmykca2ft3png_small.jpg)
![酶 _RNA 酶 抑 制 剂 （ Promega; 40U//Ltl) —脱氧核糖核酸酶I，扩 增 级 （ GibcoBRL; 1U//J) —蛋白酶 K (Boehringer Mannheim; 20 mg/ml) —M-MLV 逆 转 录 酶 （Superscript from GibcoBRL; 200U/fxl) 一大肠杆菌 DNA 聚合酶 I (Gibco-BRL; 10U/ V ) —T4 DNA 连 接 酶 （ Gibco-BRL; lU/fxl) —核糖核苷酸酶H (GibcoBRL; 2.2U//J ) —T7 RNA 聚 合 酶 （ Gibco-BRL; 50U//J ) — T叫 DNA 聚 合 酶 （ Gibco-BRL; 5U//J ) 引物 —Olig。 （ dT)18T 7 cDNA合 成 引 物 （10 ng/Ml) (5，-TAATACGACTCACTATAGGGCTTTTTTTTTTTTTTTTTT-3,) —随机六核苷酸引物（10 ng/yl) C-NNNNNN-S') —DD-PCR (dT) -锚 定 引 物 （150 ng/必 [S^TTTTTTTTTTTT (A/C/G) A-3^ S^TTTTTTTTTTTT (A/C/G) 03^; S'-TTTTTTTTTTTT (A/C/G) G-3'; S'-TTTTTTTTTnT (A/C/G) T-3'] —DD-PCR随机十核苷酸引物（1 7 ng/fxl) (26 条随机十核苷酸弓丨物的应用参见Bauer et al. 1993)](http://img.dxycdn.com/trademd/upload/userfiles/image/2016/07/A1468375086831wbht64czvnpng_small.jpg)