Differences

This shows you the differences between two versions of the page.

--- interactome:trizol [2019/12/05 11:20]
dan
+++ interactome:trizol [2019/12/05 11:51]
dan
@@ Line 25: / Line 25: @@
-  * Freshly prepared DNaseI solution included in the kit
+####Input sample requirements
-^  Check  ^  Reagent             ^   ^  1 reaction (µL)  ^  _ _ _ _ reaction (µL)  ^ Memo                                              ^
+    IMPORTANT!
-|         |  ddH2O (RNAse free)  |   |  87.5                 |                         |                                                   |
+    Perform RNA isolation immediately after sample collection or quick-freeze samples immediately after collection and store at –80°C or in liquid nitrogen until RNA isolation.
-|         | 10x DNase Buffer     |   |  10               |                         |                                                   |
-|         |  DNaseI              |   |  2.5          |                         |    12 units / µL                                               |
-^         ^  Total volume (µL)   ^   ^  100              ^                         ^  _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _  ^
+^ Sample type                ^ Starting material per 1 mL of TRIzolTM Reagent                                                  ^
+| Tissues[1]                 | 50 ー 100 mg of tissue                                                                             |
+| Cells grown in monolayer   | 1 × 10xx5 ー 1 × 10xx7 cells grown in monolayer in a 3.5–cm culture dish (10 cm2)                  |
+| Cells grown in suspension  | 5 ー 10 × 10xx6 cells from animal, plant, or yeasty origin or 1 × 10xx7 cells of bacterial origin  |
+|                            | [1] Fresh tissues or tissues stored in RNAlaterTM Stabilization Solution (Cat. No. AM7020).     |
+|                            |                                                                                                 |
+|                            |                                                                                                 |
+|                            |                                                                                                 |
+####Procedural guidelines
+  *Perform all steps at room temperature (20–25°C) unless otherwise noted.
+  *Use cold TRIzolTM Reagent if the starting material contains high levels of RNase, such as spleen or pancreas samples.
+  *Use disposable, individually wrapped, sterile plastic ware and sterile, disposable RNase-free pipettes, pipette tips, and tubes.
+  *Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin; change gloves frequently, particularly as the protocol progresses from crude extracts to more purified materials.
+  *Always use proper microbiological aseptic techniques when working with RNA.
+  *Use RNaseZapTM RNase Decontamination Solution (Cat.no. AM9780) to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.
 \\
-\\
 \\
-\\
+#### Protocol
+==1  Lyse samples and separate phases==
+  *Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.
+Cell grown in monolayer:
+  *Remove growth media.
+  *Add 0.3–0.4 mL of TRIzolTM Reagent per 1 × 105—107 cells directly to the culture dish to lyse the cells.
+  *Pipet the lysate up and down several times to homogenize.
+**Note: The sample volume should not exceed 10% of the volume of TRIzolTM Reagent used for lysis.**
+    STOPPING POINT
+    Samples can be stored at 4°C overnight or at – 20°C for up to a year.
+  *(Optional) If samples have a high fat content, centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear supernatant to a new tube.
+  *Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex.
+  *Add 0.2 mL of chloroform per 1 mL of TRIzolTM Reagent used for lysis, then securely cap the tube.
+  *Incubate for 2–3 minutes.
+  *Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.
+  *The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase.
+  *Transfer the aqueous phase containing the RNA to a new tube. Transfer the aqueous phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.
+    IMPORTANT!
+    Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase.
+  *Proceed directly to “Isolate RNA“
 \\
-\\
 \\
+==2  Isolate RNA==
+Precipitate the RNA
+  *(Optional) If the starting sample is small (<106 cells or <10 mg of tissue), add 5–10 μg of RNase-free glycogen as a carrier to the aqueous phase.
+Note: The glycogen is co-precipitated with the RNA, but does not interfere with subsequent applications.
+  *Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzolTM Reagent used for lysis. Incubate for 10 minutes.
+  *Centrifuge for 10 minutes at 12,000 × g at 4°C.
+  *Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.
+  *Discard the supernatant with a micropipettor.
 \\
+\\
+Wash the RNA
+  *Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzolTM Reagent used for lysis.
+Note: The RNA can be stored in 75% ethanol for at least 1 year at –20°C, or at least 1 week at 4°C. Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C.
+  *Discard the supernatant with a micropipettor.
+  *Vacuum or air dry the RNA pellet for 5–10 minutes.
+    IMPORTANT!
+    Do not dry the pellet by vacuum centrifuge. Do not let the RNA pellet dry, to ensure total solubilization of the RNA. Partially dissolved RNA samples have an A230/280 ratio <1.6.
+\\
+\\
+Solubilize the RNA
+  *Resuspend the pellet in 20–50 μL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting up and down.
-#### Protocol
+    IMPORTANT!
+    Do not dissolve the RNA in 0.5% SDS if the RNA is to be used in subsequent enzymatic reactions.
-==1  Sample preparation==
+  *Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.
-```
-First step of sample preparation differs if you are using (A)culture cells OR (B)Tissue specimen.
-Buffers are highlighted in blue to prevent mistakes.
-```
+\\
+Proceed to downstream applications, or store the RNA at –70°C.
+\\
+\\
+\\
+ --- //[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/12/05 11:39//