TIMING 5 hours for adults or P6 pups, and 7 hours for embryos
1| Weigh each mouse and calculate volume of the prepared 4TU solution required to deliver 400 mg/kg (e.g. 200 μL of a 50 mg/mL 4TU solution for a 25 g mouse). Lower 4TU doses generate less labeling; higher doses are not practical and potentially toxic.
2| Inject 4TU intraperitoneally using a tuberculin syringe and a 27 G needle.
3| Allow at least 4 hours of 4TU exposure prior to tissue harvesting for adults and pups and 6 hours for pregnant females.
CRITICAL STEP Exposure time can be varied depending on the experiment being performed. Labeling can be detected in as little as 2 hours following 4TU exposure to postnatal mice, but with considerably reduced yield. We have not defined the minimum exposure period for embryonic studies. We observe decreased labeling when waiting longer than 12 hours after 4TU injection.
4| Dissect out the organ or tissue of interest, transfer immediately into a 1.5 mL microcentrifuge tube, and flash freeze in liquid nitrogen. Continue with RNA preparation or store at -80°C.
PAUSE POINT Frozen tissue can be stored at -80°C indefinitely.
TIMING 2-3 hours
CRITICAL From this point on, care should be taken to maintain RNase-free conditions. Benchtop surfaces should be thoroughly cleaned, RNase-free tubes and tips used, and gloves worn at all times.
5| Precool the centrifuge to 4°C.
6| Homogenize tissue in a 1.5 mL tube using a Kontes pestle and 500 μl TRIzol per 100 mg tissue, until completely solubilized.
!CAUTION TRIzol is toxic and should be handled in a fume hood. Gloves and a lab coat should be worn.
7| Add 500 μL of additional TRIzol and vortex. Incubate 5 minutes at room temperature (approximately 22°C).
8| Add 200 μl chloroform and vortex 15 seconds. Incubate for 2-3 minutes at room temperature.
!CAUTION Wear gloves and use a fume hood when working with chloroform.
CRITICAL STEP The extended chloroform vortex is important for high RNA yield and quality.
9| Centrifuge at 12,000 g for 15 minutes at 4°C.
10| Transfer the upper aqueous phase to a new tube and add 500 μL isopropanol. Incubate for 10 minutes at room temperature.
11| Centrifuge at 20,000 g for 10 minutes at 4°C.
12| Decant liquid and wash with 1 mL 75% (vol/vol) EtOH.
13| Centrifuge at 20,000 g for 5 minutes at 4°C. Decant liquid.
14| Centrifuge at 20,000 g for 2 minutes to remove excess EtOH. Carefully pipette remaining liquid away from pellet and place tube upside down on a laboratory tissue for 2-3 minutes to dry.
CRITICAL STEP Do not over dry the RNA because this will make it difficult to re-solubilize.
15| Resuspend the pellet in 50-100 μL of RNase-free H2O. Dilute the RNA to a concentration of approximately 200 ng/μL. The RNA concentration should be determined by spectophotometry (e.g. using a NanoDrop) or with a Qubit fluorometer (see step 17).
16| Treat the RNA with DNase to remove residual genomic DNA contamination. We recommend using TURBO DNase following the manufacturer's protocol.
17| Dilute 2 μL of the total RNA in 98 μL of RNase-free H2O. Quantify the RNA concentration using a Qubit RNA BR Assay kit and a Qubit system following the manufacturer's directions.
18| Determine the integrity of the RNA samples using an Agilent Bioanalyzer. The RNA integrity number (RIN) should be > 8.0.
CRITICAL STEP RNA should be aliquoted to avoid repeated freezing and thawing.
PAUSE POINT (End Day One) RNA should be frozen and stored at -80°C overnight or until ready to proceed. Full paper
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