TRIzol™ Reagent

SDS 2.3ソフトウェアを起動すると、"user name"と"password"の入力画面が展開します。ディホルトで設定されている"user name"は、"administrator"です。"password”については、何も入力せず空欄で設定されていますので、ご利用の場合は、そのまま"OK"をクリックして頂ければ、SDS 2.3ソフトウェアは起動されます。ユーザー・アカウントを作成したい場合は、"TOOLS"メニューから"LOCAL USER ACCOUNT MANAGER"をクリックしてください。なお"LOCAL USER ACCOUNT MANAGER"の詳細情報については、SDS 2.3ソフトウェア上の"HELP"のプルダウンメニューを選択し、SDS 2.3 Online Helpで確認してください。

Degraded RNA can cause an increased absorbance at 260 nm.

Find additional tips, troubleshooting help, and resources within ourRNA Sample Collection, Protection, and Isolation Support Center.

This is most likely polysaccharides or cell membranes; DNA should be in the interphase. In samples containing blood (e.g., liver), a red viscous layer may be visible on top of the pellet. This is most likely due to blood products and should not be carried over with the supernatant.

- This is common with skin samples. It is assumed that there is fat in these samples, and the fat micelles float during the centrifugation. In skin samples, the micelles pick up melanin pigment and cause the aqueous phase to appear colored. Fat micelles may also pick up pigment from the TRIzol Reagent itself and cause a pinkish color. If a sample is thought to contain fat, the sample homogenate in TRIzol Reagent may be centrifuged prior to addition of chloroform. The fat will appear as a clear layer at the top of the supernatant; this should be pipetted off and discarded.
- If a sample contains a lot of blood, the aqueous phase may appear cloudy and/or yellowish (this may be due to iron in the hemoglobin). If the centrifuge used is not cold, the organic phase will be a deeper maroon color; some of this color may come into the aqueous phase and cause it to appear orange or yellow.
- A pinkish aqueous phase may also be caused by overdilution of the sample (i.e., a sample to TRIzol Reagent ratio > 1:10), as well as too much salt or protein in the sample. This can cause premature phase separation, which can be remedied by adding a bit more TRIzol Reagent to the sample. If the RNA is isolated from a pinkish aqueous phase, chances are that it will be contaminated with DNA.

These are our recommendations:

1. Upstream tissue procurement and tissue specimen preparation—if possible, fix tissues within one hour of surgical resection. Extensive degradation of RNA can occur before completion of the fixation process. The optimal fixation time is 12-24 hours, using neutral-buffered formalin or paraformaldehyde. Fixed tissues should be thoroughly dehydrated prior to the embedding process.
2. Block storage—storage of blocks without cut faces, when possible, prevents ongoing damage from exposure to atmospheric oxygen, water, and other environmental factors such as light and infestation (fungus, insects, etc.).
3. Choice of tissue type, size, and amount being used for RNA isolation—the recommended tissue thickness is 10-20 µm. The number of sections used is determined by the tissue type (which impacts cell density) and surface area (recommended size: 50-300 mm2). Excess starting material can cause filter clogging, resulting in poor yield.
4. Avoid using an excessive amount of paraffin for embedding tissues—when possible, excess paraffin should be trimmed away prior to starting the purification protocol. For xylene-based purification methods, two xylene treatments at room temperature should be sufficient for complete deparaffinization. If desired, you can perform a more rigorous 37-55 degrees C treatment for up to 30 minutes. After the xylene deparaffinization, it is crucial that the 100% ethanol is completely removed and the pellets are dry after the two 100% ethanol washes. The magnetic bead method employs novel chemistries to deal with the paraffin that limits input to 20 µm sections.

Read more about RNA isolation from FFPE tissues here (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-sample-extraction/working-with-ffpe-samples.html).