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qRT-PCR with Standard Curve

    Clean your bench and pipetman with RNase Away.
    Anywhere below that says H2O means DEPC H2O.
    For each gene you are testing, you will also do:

    1. a standard curve using "bulk" RNA from either WT cells (29-13, e.g.) or -tet cells.
    2. an H2O control containing primers, SYBR green, and H2O.
    3. a -RT control. Do an -RT for each sample (e.g., -tet and +tet), but it is OK to do it only on the housekeeping gene (e.g., B-tubulin) and not the gene of interest.
    DNase Treatment: with Ambion DNaseI DNAfree kit
    • 10μg RNA
    • 5μl 10X DNaseI buffer
    • 1μl rDNaseI
    • 0.5μl RNase inhibitor
    • H2O to 50μl

    Incubate at 37°C 30 min, add 1μl rDNaseI, and incubate 37°C 1 hr. Add 150μl H2O followed by 200μl phenol/chloroform/IAA (acidic pH). Spin 25 min at 4°C and take top layer. Precipitate RNA with 20μl 3M NaOAc + 1μl glycogen + 800μl cold 100% EtOH in -20°C overnight. Spin down RNA at 13,000rpm 30 min, remove supernatant, dry pellet 10 min, and resuspend pellet in 20μl DEPC H2O. Spec RNA.

    Reverse Transcription Reaction:

    There are two different kits/protocols that can be used. Applied Biosystems Taqman kit (Brianna) or BioRad iScript kit (Kyle). Both are explained below.

    With Applied Biosystems Taqman kit
    • 4μg RNA per reaction
    • 5μl Taqman RT buffer
    • 11μl 25mM MgCl2
    • 10μl dNTP mix
    • 3.5μl Random Hexamers
    • 0.5μl RNase Inhibitor
    • 1.5μl Multiscribe RT
    • H2O to 50μl

    Incubate at 25°C for 10 min, 48°C for 45 min, 95°C for 5 min, cool on ice, then add 1.5μl RNaseH (Ambion/Applied Biosystems), incubate at 37°C for 20 min, then at 65°C for 20 min.

    With BioRad iScript cDNA synthesis kit
    • 1μg RNA per reaction
    • 4μl 5X iScript reaction mix
    • 2μl primer (oligo d(t), random hexamer, etc.)
    • 1μl iScript RT
    • Depc H2O to 20μl

    Incubate at 42°C for 90 mins, 85°C for 5 mins, hold at 12°C

    Q-PCR Setup

    Assume a 1:1 conversion of RNA into cDNA. For example, if you started with 4μg of RNA and ended up with 50μl of cDNA, then you assume cDNA = 80ng/μL.

    • Bulk cDNA:
      • - RT dilute 80ng/μL at 1:5 = 16ng/μl
      • + RT serial dilute
        • 1X = 1:5 dilution from 80ng/μL = 16ng/μL
        • 1/4X = 1:4 dilution from 16ng/μL = 4ng/μL
        • 1/16X = 1:4 dilution from 4ng/μL = 1ng/μL
        • 1/64X = 1:4 dilution from 1ng/μL = 0.25ng/μL
        • 1/256X = 1:4 dilution from 0.25ng/μL = 0.0625ng/μL
        • 1/1024X = 1:4 dilution from 0.0625ng/μL = 0.015625ng/μL

    • Sample cDNA:
      • Dilution for gene of interest use 1:20 dilution of sample RT at 80ng/μL = 4ng/μL
      • Dilution for housekeeping (control) gene use 1:20 dilution of sample RT at 4ng/μL = 0.2ng/μL

    • Primers (all primer stocks at 100μM):
      • Add 15μL of each forward and reverse primers to tube and dilute with 970μL H2O, primer mix = 1.5μM

    Following plate map (example in Q-PCR plate map file) add 2.5μL of cDNA to each well. Mix 1.5μM primers with SYBR green Master Mix in a tube (10μl primers to 12.5μl SYBR green per well for as many wells as needed) and add 22.5μL of primer/SYBR mix to each well. Cover plate with adhesive cover, use squeegee to press down cover, and place in machine.

    Use program:
    • Cycle 1, Step 1 = 50°C 2 min
    • Cycle 2, Step 1 = 95°C 10 min
    • Cycle 3 40X, Step 1 = 95°C 15 sec, Step 2 =60°C 1 min (take measurement)
    • Cycle 4 80X, Step 1 melt curve = 55°C 10 sec, then temp change in 0.5°C increments to 94.5°C (take meas.)

    Download Protocol: Q-PCR Protocol, Q-PCR Plate Map
    (note requires Microsoft Word to open)