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--- interactome:trizol [2019/12/05 11:32]
dan
+++ interactome:trizol [2019/12/05 11:51] (current)
dan
@@ Line 22: / Line 22: @@
 | RNase-free water of 0.5% SDS         |       |
 | (Optional) RNase-free glycogen       |       |
 ####Input sample requirements
-**IMPORTANT!**
+    IMPORTANT!
     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.
@@ Line 45: / Line 40: @@
 |                            |                                                                                                 |
 |                            |                                                                                                 |
@@ Line 58: / Line 50: @@
   *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.
-\\
-\\
-\\
 \\
 \\
@@ Line 69: / Line 58: @@
   *Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.
-'''
-Tissues:
-  *Add 1 mL of TRIzolTM Reagent per 50–100 mg of tissue to the sample and homogenize using a homogenizer.
-'''
 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.
-'''
-Cells grown in suspension:
-  *Pellet the cells by centrifugation and discard the supernatant.
-  *Add 0.75 mL of TRIzolTM Reagent per 0.25 mL of sample (5– 10 × 106 cells from animal, plant, or yeasty origin or 1 ×107 cells of bacterial origin) to the pellet.
-**Note: Do not wash cells before addition of TRIzolTM Reagent to avoid mRNA degradation.**
-  *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.
+    IMPORTANT!
+    Do not dissolve the RNA in 0.5% SDS if the RNA is to be used in subsequent enzymatic reactions.
+  *Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.
+\\
+Proceed to downstream applications, or store the RNA at –70°C.
+\\
+\\
+\\
+ --- //[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/12/05 11:39//