Molecular Methods in Developmental Biology Molecular Methods in ...

44 Steinbach and Rupp1. Transfer embryos or tissue explants to autoclaved microfuge tubes (1.5 mL)and carefully remove excess buffer. Avoid shearing or air contact of specimen,in particular with tissue explants, as this may result in instant cell lysis andRNA degradation.2. Add 1 mL per 50–100 mg tissue of Tristar solution, according to themanufacturer’s instructions. For Xenopus, we use 200 µL per 3–5 embryos, or100 µL per 3–5 tissue explants.3. Add the same amount of Tristar solution to a separate tube, which is carriedthrough the whole procedure as a mock RNA sample (see Note 3).4. Vortex for 10 s, incubate for 5 min at room temperature and freeze at –70°C(although it is possible to proceed directly, freezing improves tissue lysis).Samples can be stored at this point for months, if required.5. Thaw lysates at room temperature and vortex briefly.6. Add 0.2 volumes of chloroform and vortex for 15 s.7. Centrifuge samples (14,000g, room temperature, 5 min). The emulsion will separateinto three phases. Transfer the upper, aqueous phase with the RNA to anautoclaved microfuge tube; avoid the interphase and the lower, organic phase,which contain genomic DNA and protein.8. Add 0.5 starting volumes of isopropanol–tRNA solution. Mix by inverting thetubes several times.9. Incubate for 15 min at room temperature.10. Centrifuge (14000g, 4°C, 10 min).11. Carefully remove the supernatant.12. Add 500 µL cold 70 % EtOH and centrifuge (14,000g, room temperature, 5 min).Discard the supernatant, be careful to retain the RNA pellet.13. Air dry the precipitated RNA for approximately 10 min at room temperature.Cover the open microfuge tubes (e.g., with lint-free paper wipes) to avoid airbornecontamination.14. Dissolve the RNA in DEPC–H 2 O at a concentration of approximately 0.1–0.5µg/µL. (One Xenopus embryo [prehatching stages] yields approximately 2–5 µgof total RNA; we add 25 µL DEPC–H 2 O per embryo, or 2 µL DEPC–H 2 O pertissue explant.)15. Vortex and incubate for 10 min at 56°C. Collect the liquid by brief centrifugation.Store the RNA at –70°C.3.2. RT ReactionIn this step, a single-stranded cDNA library is generated from total cellularRNA using short, random oligonucleotides and reverse transcriptase (RTase).Thus, each sample can be analyzed for the abundance of multiple mRNAs,including reference mRNAs, which is an important aspect for quantitativeanalysis (see Subheading 3.4.). We find that random hexamer oligonucleotidesoutperform oligo-dT as primers. Reverse transcription of some mRNAs (e.g.,Xenopus MyoD [XMyoD]) is very temperature sensitive, presumably due to