点赞

收藏

分享

RNA isolation protocol: cells in culture

互联网2013-11-13

578

实验步骤

1. Using at least 10⁶ cells, aspirate off the media and wash X1 with ice cold PBS (1-2 ml)

2. Aspirate off the PBS (Remove as much as possible) and add 1 ml of trizol

3. Scrape the plate briefly, then remove the trizol with a pipette and deposit the trizol/cell lysate into a 1.5 ml eppendorf tube.

4. Let sit for 5 min at room temperature.

5. Add 250 μl of chloroform and shake the tube vigorously for about 15 seconds.

6. Let sit for 5 min at room temp.

7. Centrifμge at 10,000 rpm for 5 min.

8. At this point, there will be three layers in each tube:
    1) Top layer: clear – aqueous
    2) Middle layer / interphase - white precipitated DNA
    3) Bottom layer – pink organic phase

9. Carefully pipette off the aqueous phase. With the larger pipette, it is harder to control the rate and force of fluid withdrawal and this increases the likelihood of drawing some of the organic or DNA phase. Leave behind some of the aqueous phase (about 1 mm above DNA layer to prevent DNA contamination). Place in another 1.5 ml eppendorf.

10. Add 550 μl of isopropanol to the aqueous phase and mix gently. Let this sit at room temp for 5 min.

11. Centrifuge at maximal speed (14,000 rpm) for 20 min. If a low yield is expected, centrifuge for 30 min.

12. Remove and place on ice. There should be a pellet barely visible at the base of each tube. Pour off the isopropanol and add 1 ml of 75% EtOH in DEPC treated H₂ 0. Mix gently. Recentrifuge at 9500 rpm for 5 min.

13. Pour off the EtOH and let the pellets air-dry.
This is a critical step. If the pellets dry out too long or too much, the RNA crystallizes and is very difficult to resolubilize. If not enough of the EtOH evaporates, this also prevents the RNA from going into solution. After pouring off the bulk of the EtOH wash, there will be approx 30-40 μl left in the bottom of the eppendorf. To quicken the evaporation, centrifuge the tubes briefly to force remaining fluid on the side of the tube to the bottom, then pipette off as much of the EtOH as is feasible. The best time to add DEPC treated water to the RNA pellet is when there is only a tiny meniscus of solution left around the pellet itself.

14. Add approx 15-25 μl (depending on yield) of either DEPC treated TE buffer or water to the RNA pellet. To a small eppendorf tube, dilute the RNA 1/40 (1.2 μl in 48.8 μl of TE buffer) and add to a microcuvette (path length = 1 cm). Then measure the absorbance at 260 nm. The 260/280 ratio should be greater than 1.8. If less than 1.5-1.6 or so, the RNA is likely, at least partially degraded. Lower ratios also suggest DNA or thiocyanate contamination. The concentration is essentially the equivalent of the OD at 260 nm (in μg/μl).

The only difference is the first step. Add 1 ml of trizol to a sterile culture tube (preferably 12x75 mm). To this tube, add the frozen tissue (try not to add more than approx 20 mg). On ice, pulverize the tissue with the homogenizer at a setting of 25 out of 30 for a total of 10 sec x2. Then pour the trizol solution into a 1.5 ml eppendorf and proceed as above.

DNAse treatment:

1. The DNAse cocktail consists of the following (per sample):

    RQ1 Rnase free Dnase: 1 μl
   Dnase 10x reaction buffer 2 μl
   DEPC-treated H₂ O 6 μl
   Rnase Out 0.5 μl

2. Make a master mix of the above based on the number of RNA samples being treated.

3. Prepare the RNA in the following way:

Add 2 μg of RNA (calculated by 2 μg/the concentration in μg/μl) to a small eppendorf Bring the total volume of the RNA to 11 μl by adding additional DEPC treated water. (For example, if your RNA concentration is 1 μg/μl, add 2 μl of the RNA to 9 μl of DEPC water).

Add 9 μl of the Dnase master mix to the RNA bringing the total volume to 20 μl

4. This step should be done in a thermal cycler. Incubate the samples at 37°C for 15 min, followed by 65°C for 20 min, then place on ice. Briefly centrifuge each sample to assure all of the volume lies in the bottom of the tube. The DNAase treated RNA can then be used immediately for the reverse transcription reaction.

Reverse transcription of DNAse treated RNA:

1. Under most circumstances, each sample of RNA (1 μg or 10 μl from the DNase treatment reaction) will be run with reverse transcriptase with the second 1 μg aliquot being used for a no-RT control. On the other hand, the DNase treatment protocol is 100% effective and has been so for years. Lately, I’ve been using only a few samples for no- RT control for a given set of samples.

2. For each sample, you’ll mix together the following:

    13 μl DEPC treated water
   16 μl 5X first strand buffer
   7 μl DTT (0.1 M)
   8 μl random primers (concentration = 0.1 μg/μl or a 1/30 dilution of the 3 μg/μl)
   8 μl BSA
   3 μl dNTPs
   1 μl RNAse Out

The total from the above reagents is 56 μl. Mix by vortexing.

3. Aliquot 28 μl each into two separate 0.5 ml eppendorf tubes. One tube will serve as the RT reaction (to which 2 μl of the MMLV reverse transcriptase is added) and the other, the no-RT control, to which 2 μl of H₂ O is added.

4. To each tube, add 10 μl of the DNase treated RNA from above.

5. Mix well by pipetting.

6. Incubate all samples at 37°C for 1 hour, then 95°C for 5 min. The latter step fully inactivates remaining enzymes (i.e. MMLV RT and any remaining Dnase).

7. Use immediately for PCR or store at –20°C. If this is the end of the day, you can leave in the thermal cycler overnight at 4° and store in the –20°C first thing the next day.

8. An alternate approach to the above might include the following:

One needs a total 28 μl of the non-RT containing master mix per sample. Randomly select two or three samples from a group and prepare no-RT controls by aliquotting out 28 μl per sample into 0.5 ml tubes and adding 1 μg of RNA to each along with an additional 2 μl of DEPC treated H₂ O (you can skip this if you want).

To the remaining RT master mix, add 2 μl of MMLV RT/sample being prepared, mix by vortexing, and then aliquot 30 μl per tube for each sample (for example, if you have a total of 10 samples, you will reverse transcribe all 10, but want 2 or 3 samples additionally for no RT controls to assure that the Dnase treatment step worked. Thus, you would multiply the above master mix x 7 or 8. 1x of the master mix is for 2 samples.

Thus, if you’ll need a total of 12 reactions (for both RT and no-RT reactions) then you will need 6x of the master mix cocktail. Once made, aliquot off the two no-RT samples (28 μl each), add 20 μl of the MMLV RT (2 μl/sample) to the master mix. Vortex the mix, then aliquot 30 μl per tube for 10 tubes. To each, add the corresponding 1 μg (10 μl) of RNA and incubate as above.

相关产品推荐

mRNA纯化试剂盒|高效、灵敏、高性价比| mRNA Isolation Master Kit

mRNA纯化试剂盒|高效、灵敏、高性价比| mRNA Isolation Master Kit

￥2600

esxB/esxB蛋白Recombinant M_y_c_obacterium t_u_b_erculosis ESAT-6-like protein EsxB (esxB)重组蛋白10KDA culture filtrate antigen CFP-10 Secreted antigenic protein MTSA-10蛋白

esxB/esxB蛋白Recombinant M_y_c_obacterium t_u_b_erculosis ESAT-6-like protein EsxB (esxB)重组蛋白10KDA culture filtrate antigen CFP-10 Secreted antigenic protein MTSA-10蛋白

￥2328

AQP1/AQP1蛋白/Aquaporin-CHIPUrine water channelWater channel protein for red blood cells and kidney proximal tubule蛋白/Recombinant Human Aquaporin-1 (AQP1), partial重组蛋白

AQP1/AQP1蛋白/Aquaporin-CHIPUrine water channelWater channel protein for red blood cells and kidney proximal tubule蛋白/Recombinant Human Aquaporin-1 (AQP1), partial重组蛋白

￥69

KTO: KIT A643 VALIDATION PROTOCOL (L)-Anatel A643a Accessories

KTO: KIT A643 VALIDATION PROTOCOL (L)-Anatel A643a Accessories

￥50094

RNA Easy Fast植物组织RNA快速提取试剂盒（DP452）

RNA Easy Fast植物组织RNA快速提取试剂盒（DP452）

￥1080

相关问答

问

WB显出膜没有条带

问

细胞增殖

问

2小时能完成Western Blot实验，可能吗？

相关方法

使用 Trizol 纯化 RNA 实验

2024-05-14

利用 Marligen 总 RNA 快速纯化系统纯化总 RNA 实验

2024-05-14

用T4 RNA 连接酶连接 RNA 分子

2024-05-14

推荐阅读

RNA Isolation Protocol

Total RNA isolation protocol

RNA Isolation Protocol from LCM Samples

提问

扫一扫

丁香实验小程序二维码

实验小助手

丁香实验公众号二维码

关注公众号

反馈

TOP

打开小程序