2007-10-31
org.kosen.entty.User@e22958
진정숙(jinpaldook)
- 5
RNA를 혈관과 신장에서 뽑으려고 하는데 3번을 trizol method, QIAGEN method 로 뽑아 보내줘도 딱히 질이 좋지 않다는데 혹 RNA만 전문적으로 뽑아주는 곳은 없을까요?
- RNA
지식의 출발은 질문, 모든 지식의 완성은 답변!
각 분야 한인연구자와 현업 전문가분들의 답변을 기다립니다.
각 분야 한인연구자와 현업 전문가분들의 답변을 기다립니다.
답변 5
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답변
유용규님의 답변
2007-10-31- 0
>RNA를 혈관과 신장에서 뽑으려고 하는데 3번을 trizol method, QIAGEN method 로 뽑아 보내줘도 딱히 질이 좋지 않다는데 혹 RNA만 전문적으로 뽑아주는 곳은 없을까요? 얼마전에 takara에서 RNA추출용 키트가 나온것 같던데 Eukaryotic cell에서 추출하는 용도더군요 추출효율과 순도가 높다는말을 들은적이 있는데..... 문의해보시는건 어떠실지요 -
답변
김남철님의 답변
2007-10-31- 0
>RNA를 혈관과 신장에서 뽑으려고 하는데 3번을 trizol method, QIAGEN method 로 뽑아 보내줘도 딱히 질이 좋지 않다는데 혹 RNA만 전문적으로 뽑아주는 곳은 없을까요? 보내셨다는 곳에서는 뽑아주지 않나요? 제가 있는 곳의 facility에서도 그냥 trizol method를 씁니다. 자기들이 여러가지 다 써봤는데 경험상 그게 제일 낫다는군요.. 샘플은 신선한 것인가요... 원래 조직에서 뽑으면 셀에서 뽑은 것보다는 조금 드럽게 나오는데 그것도 감안한 것인가요... -
답변
전현주님의 답변
2007-11-01- 0
>RNA를 혈관과 신장에서 뽑으려고 하는데 3번을 trizol method, QIAGEN method 로 뽑아 보내줘도 딱히 질이 좋지 않다는데 혹 RNA만 전문적으로 뽑아주는 곳은 없을까요? 다음 방법을 한번 사용해 보세요 저도 몇번 실패하고 나서 겨우 얻은 protocol인데 결과가 좋았어요. OD230값이 잘 안나왔었는데 그건 phenol과 ethanol때문이라는군요. 마지막 ethanol제거할 때 뚜껑을 열고 오래 돌리는 것이 핵심인 것 같습니다. TRIZOL만 쓰면 230 값이 잘 안나오는데, 저도 전에 TRIZOL만 가지고 한 것으로 micro array한 적도 있거든요. 회사에 따라 사람에 따라 워낙 비싼 실험이라 까다롭게 구는 사람도 있더라구요. 그럼 이번에는 성공하시기를.... Total RNA Isolation Using Trizol and Qiagen RNAeasy Columns Overview This protocol is quick and works very well for preparing 20 to 40 ug of very clean, salt free, RNA. The RNA prepared from this protocol is ready for target preparation using the Ambion Message Amp II procedure to produce aminoallyl labeled cRNA. Materials Required RNAase-free mortar and pestle: cover the mortar and pestle with aluminum foil and bake at least 3 hours at 180°C RNAase-free 1.5 or 2.0 mL (preferred) microfuge tubes Liquid nitrogen Microfuge RNAase-free pipette tips Qiagen RNAeasy Mini elute columns and buffers (Qiagen Cat # 74204) DEPC-treated H2O Trizol (Invitrogen) Procedure 1. Homogenize tissue in liquid nitrogen. It is not necessary to homogenize large amounts of tissue as the Qiagen RNAeasy columns can only bind ~40 ug of RNA and overloading the column is not advised. If you are working with pooled samples you may find that you have more ground sample than you can use. It is convenient to have a small measuring device (teaspoon, grooved spatula, etc) to transfer the ground material directly to the Trizol in the microfuge tube. a. Chill mortar with ~100 mL of liquid nitrogen. b. Add frozen tissue after nitrogen is nearly completely evaporated. c. Grind tissue quickly but carefully. d. When liquid has fully evaporated, grind faster to produce a fine talc-like powder. 2. Add 1/8 to 1/4 teaspoon to 1.0 ml of Trizol. It is important to mix well immediately by vortexing and not allow tissue to thaw that is not in contact with the Trizol. You may want to prewarm the Trizol (35-40 C) so that it does not freeze when it comes into contact with the frozen tissue. This is the most critical step in the procedure as once the tissue is completely mixed with the Trizol, it is protected from RNAases. 3. Incubate for five minutes at room temperature (RT), votexing frequently. 4. Add 0.2 ml of chloroform to the Trizol, and vortex for 15 seconds. 5. Incubate for 1 minute at RT, vortex again for 15 seconds. 6. Centrifuge at 15,000 xg for 10 minutes to separate phases 7. Remove 200 ul from the top layer and add to 700 ul of Qiagen RLT buffer in a new tube. Remove the rest of the top layer and freeze at -20 to serve as a backup in case your initial yield is low. 8. To the 200 ul of sample now combined with 700 ul RLT buffer, add 500 ul of 96-100% ethanol. Mix well by vortexing but do not centrifuge. 9. Apply half of your sample (~700 ul) to a Qiagen MinElute spin column placed in a 2 ml microfuge tube. Spin 15 seconds at ~10,000 rpm. Discard flow through and repeat procedure with the second half of your sample. 10. Remove the Minelute column to a new 2 ml microfuge tube and add 500 ul of RPE to the spin column. Spin 15 seconds at ~10,000 RPM. Discard flow through. 11. Add 750 ul of 80% ethanol and spin at ~10,000 rpm for 15 seconds. Repeat this step a second time with another 750 ul of 80% ethanol. This step is repeated to ensure removal of all guanidine salts that may inhibit downstream applications. 12. Transfer the Minelute spin column to a new 2 ml microfuge tube. Spin for 5 minutes at top speed with the cap off. This ensures the removal of trace amounts of ethanol that may interfere with downstream applications. 13. To elute RNA, transfer spin column to a new, 1.5 ml microfuge tube. Elute with 10 ul of RNAase free water. Repeat with another 10 ul of RNAase free water. If you suspect low RNA concentration, you may elute with 12 ul of RNAase free water. It is desirable to have a concentration of ~ 1 ug/ul if possible. Check concentration on a gel or spectrophotometer. If your initial yield from the 200 ul is low, you may want to consider precipitating the remainder of your sample with an equal volume of isopropanol and resuspending in 200 ul of H2O for concentration using a minelute RNAeasy column. -
답변
손은정님의 답변
2007-11-01- 0
>RNA를 혈관과 신장에서 뽑으려고 하는데 3번을 trizol method, QIAGEN method 로 뽑아 보내줘도 딱히 질이 좋지 않다는데 혹 RNA만 전문적으로 뽑아주는 곳은 없을까요? Genocheck에서는 RNA prep부터 해주는 것으로 알고 있습니다. 저희는 그냥 RNA는 저희가 뽑아서 드리긴 했지만 RNA 추출도 해주는 것을 들었습니다. 한번 직접 genocheck에 여쭤보시면 어떨까합니다. -
답변
전현주님의 답변
2007-11-01- 0
>RNA를 혈관과 신장에서 뽑으려고 하는데 3번을 trizol method, QIAGEN method 로 뽑아 보내줘도 딱히 질이 좋지 않다는데 혹 RNA만 전문적으로 뽑아주는 곳은 없을까요? 광고 메일이긴 한데 다음 정보를 오늘 아침에 받아서.... 혹시 잘 안되면 invitrogen에 한번 찾아보세요. More ways to get intact RNA Invitrogen RNA purification We offer more ways to isolate RNA, so you can rely on us for the quality and purity you need for your research. Choose your format: 1) The original?TRIzol® Reagent The most cited RNA purification reagent, exclusively from Invitrogen Great for difficult samples The familiar spin column?PureLink™ Micro-to-Midi™ Total RNA Isolation System 2) Total RNA purification in a familiar and time-saving spin-column format Ideal for applications requiring binding of 1?1,000 ? of RNA from a single prep The best of both worlds?TRIzol® Plus RNA Purification System 3) Integrated TRIzol® Reagent and PureLink™ Micro-to-Midi™ spin-column format Recommended for downstream applications requiring the highest purity Make More Happen. Check out www.invitrogen.com/rnapreps for our complete portfolio of RNA purification products. Your purchase of Invitrogen’s ChargeSwitch® and PureLink™ nucleic acid purification products supports breast cancer education and research. Learn how at www.invitrogen.com/pinkribbon. Invitrogen Corporation Corporate Headquarters 1600 Faraday Avenue Carlsbad, CA 92008 As a key partner in the global life science community, Invitrogen provides products and services that support academic and government research institutions as well as pharmaceutical and biotechnology companies. Copyright ?007 Invitrogen Corporation. All rights reserved.