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    • CommentAuthorjround77
    • CommentTimeMay 25th 2010 edited
     
    • This protocol is used to test for the presence of particular mRNAs in fish embryos at a given stage of development. It is divided into 4 parts: isolation of RNA, removal of trace amounts of contaminating genomic DNA, cDNA synthesis, and PCR from the cDNA template with gene-specific primers.
    • This protocol was tested on 24-48 hpf fish embryos. More embryos may be required for younger stages, and fewer embryos for older stages.
    • This protocol can also be used to isolate RNA from Xenopus embryos.
    • TRI Reagent is highly toxic. WORK UNDER THE FUME HOOD for Steps 3-10. Discard all tubes and tips that touch this reagent in the fume hood waste bin.
    • RNA is easily degraded by RNAses present on our skin and in dust. Wear gloves, use filtered pipette tips, and use tubes and solutions known to be RNAse-free.

    PART 1: Isolation of total RNA from Embryos

    1. Remove the chorion from 20-25 embryos and place in an eppendorf tube.
    2. Remove all liquid, using a yellow tip to remove the last of the liquid between embryos.
    3. WORKING UNDER THE HOOD, add 200 ul of TRI Reagent to the tube of embryos.
    4. Mash embryos using a small plastic pestle until the solution is cloudy and you
    can’t see any more tissue. **Add 800 ul more TRI reagent and let sit at RT 5’.
    5. Centrifuge at high speed (>12,000xg) 5’ and transfer supernatant to fresh tube. Discard tube with pellet in hood waste.
    6. Then add 100 ul of 1-bromo-3-chloropropane (1B3CP). Shake vigorously for 15-20 seconds.
    7. Centrifuge at high speed for 10’.
    8. Carefully transfer upper phase to fresh tube. Do not disturb the interface. You may have to leave a bit of the upper phase to accomplish this. Discard the lower phase in the TRI waste bottle and the tube in the hood waste.
    9. Add 500 ul of 2-propanol (IPOH) **USE RNASE FREE** to upper phase then VORTEX.
    (IF CONTINUING TO DNAse TREATMENT: thaw buffer on ice)
    10. Centrifuge 10’ and discard liquid. RNA is in a white pellet in the bottom of the tube.
    11. Add 1 mL of **RNAse-free** 75% ethanol (make fresh), shake the pellet loose, and centrifuge 5’.
    12. Carefully remove liquid with a pipette and let pellet air-dry for 3-5' (can do this under the hood if ETOH is taking to long to evaporate).
    13. Re-suspend pellet in 45 ul RNAse-free water by slow pipetting with a filter tip.
    14. Store RNA at -80C or proceed to DNAse treatment with RNA on ice in order to remove trace amounts of genomic DNA.

    PART 2: DNAse treatment of isolated RNA (using Ambion Turbo DNAse Kit)

    1. Pre-warm heat block or water bath to 37C.
    2. Set up DNAse reaction *on ice* in an eppendorf tube as follows:

    45 ul RNA
    5 ul 10X Turbo DNAse Buffer
    1.5 ul DNAse (spin enzyme before using)

    3. Cap and flick tube to mix, then pool contents to bottom of tube by spinning for 5 seconds in the bench-top microcentrifuge. Incubate 30’ at 37C. With about 15' left, thaw (to slush) the DNAse inactivation reagent.
    4. Add another 1.5 ul DNAse, mix and spin as before, and incubate an additional 30’ at 37C.
    5. Add 10 ul DNAse inactivation reagent and mix by flicking tube gently.
    6. Incubate at RT 6-7’, flicking tube every minute to re-mix contents.
    7. Centrifuge 2’ and transfer upper clear phase to fresh tube on ice. This is your DNA-free RNA.
    Store at -20C or keep on ice while proceeding to cDNA synthesis.
    Note: This protocol was tested by directly proceeding to cDNA synthesis. In theory, any delay risks degradation of your RNA by RNAses, but I have not tested this directly.

    PART 3: Synthesis of cDNA from RNA (Using Phusion RT-PCR Kit available through NEB)

    1. Pre-warm heat block to 65C.
    2. Set up two eppendorf tubes *on ice*, each containing the following:

    2 ul DNA-free RNA
    1 ul dNTP mix
    1 ul OligodT primer
    6 ul RNAse-free water

    3. Incubate at 65C for 5’. Transfer contents of tubes to two small, 500-ul PCR tubes.
    4. Add the following to tube #1 (+RT):
    2 ul 10X Buffer for RT
    6 ul RNAse-free water
    2 ul Reverse Transcriptase *SPIN ENZYME BEFORE USE*

    Add the following to tube #2 (-RT):
    2 ul 10X Buffer for RT
    8 ul RNAse-free water
    **No Reverse Transcriptase

    5. Flick tubes and spin to pool contents. Run the following PCR program (JR CDNA):
    25°C for 10’, 40°C for 30’, 85°C for 5’, then hold at 4C.
    6. Store cDNA reactions at 4C until ready to proceed to PCR.

    Note: The cDNA synthesis reaction that omits RT is a very important control for any RT-PCR experiment. In the “No RT” reaction, no cDNA should be synthesized from the RNA, and therefore no PCR product should be obtained in the subsequent PCR amplification step described below. If you do get an amplification product from the “No RT” sample in your next PCR, this would indicate that your RNA prep was likely contaminated with genomic DNA. This would mean that any band you observe in the “+RT” condition is NOT a reliable indicator that your mRNA of interest is present in your total RNA. It could have been derived from the genomic DNA.

    PART 4: PCR to amplify sequence of interest from cDNA

    Set up 2 PCR reactions for each gene of interest:
    (*One using +RT cDNA, and one from the –RT reaction.)
    35.5 ul water
    10 ul 5X Phusion Buffer
    1 ul dNTP mix
    1 ul forward primer for gene of interest
    1 ul reverse primer for gene of interest
    1-2 ul cDNA (+RT or –RT)
    0.5 ul Phusion Hot Start Taq Polymerase

    Flick tube to mix and spin to pool contents. Run the following program:
    98C 30 seconds
    98C 10 seconds
    48C 10 seconds (temp varies based on primer Tm)
    72C 60 seconds
    Go to Step 2, 25 cycles
    72C 5 minutes
    4C Hold
    Run your PCR products on a 1% agarose gel. Good luck!