User:David Benjamin Nyer/Notebook/PcTF breast cancer/2016/06/06

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cDNA Synthesis from MCF7 mRNA

I'll be measuring the concentration of the RNA samples from MCF7 transfected with PcTF, then creating cDNA using the SuperScript III First-Strand Synthesis kit. See the protocol for full instructions.

Procedure

Materials

  • Heat block set to 65°C
  • Heat block set to 37°C
  • Thermal Cycler (PCR machine)
  • RNase-free 0.2 mL strip tubes, 0.5 mL tubes, 1.5 mL tubes
  • Ice bucket (additional cold block is optional)
  • SuperScript III First-Strand Synthesis kit (Life Technologies 18080-051)
  • RNA samples - see the RNA extraction protocol


Procedure

cDNA SYNTHESIS

  1. If necessary, retrieve the RNA from the -80°C freezer and thaw on ice or a cold block. If freshly made, keep the samples on ice/ a cold block.
  2. NOTEBOOK ENTRY: Measure the concentration of all RNA samples (even if you have previously measured the RNA that was retrieved from the -80°C freezer. RNA degrades over time, even at -80°C).
  3. Retrieve the SuperScript III kit from the -20°C freezer.
  4. Thaw the following at room temperature on your bench: 50 μM oligo(dT) primer, 10 mM dNTP mix, Water, 10x RT buffer, 25 mM MgCl2, 0.1 M DTT
  5. Keep the following on ice or in a cold block: SuperScript III RT, RNaseOUT, RNase H.
  6. NOTEBOOK ENTRY: In labeled, clean RNase-free 0.5 mL tubes, set up oligo(dT) Primer-RNA annealing reactions.
  7. Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.
  8. NOTEBOOK ENTRY: In a clean, RNase-free 1.5 mL tube, make enough cDNA synthesis mix for all desired reactions. Transfer 10 μL this mix into labeled, clean 0.2 mL, 8-tube PCR strip(s).
  9. Transfer each primer-RNA annealing reaction into a 10 μL aliquot of cDNA synthesis mix.
  10. Synthesize cDNA: Place the samples into the thermal cycler (PCR machine) and run the following program: 1x 50°C/ 50 min., 1x 80°C/ 5 min., 4°C/ ∞
  11. Degrade the RNA: Remove the samples from the thermal cycler. Add 1.0 μL RNase H to each sample. Mix by flicking the tubes and incubate at 37°C for 20 min.
  12. Proceed to stage B or store at -20°C. (Note: the cDNA is PCR-ready and does not need to be cleaned-up)


SAMPLE SET-UP FOR RT-PCR
HIGHLY recommended for organizing samples for downstream PCR analysis

  • Get a fresh 8-tube PCR strip. These are MUCH easier to handle and store than individual 0.5 mL tubes.
  • Use four tubes for each unique cDNA sample. Label them in a fashion similar to the example illustration below.
    • About this example: U2OS E001 is cDNA from a U2OS cell line that has been transfected with an experimental (E) transcription factor, while U2OS C001 represents the mock-transfected control (C) that was processed at the same time. Labels U2OS E002 and U2OS C002 will be used for the next experiment/ control set of cDNA. K562 E001/ C002 will be used when the cell type is changed to K562.
    • General advice: You should use a labeling scheme that takes into account different cell types (if using different ones), and the number of cDNAs you will need to produce. For a time-course on a single cell type, a series of labels such as Ctrl 001, Dy2 001, Dy4 001, Dy6 001 could be used to label cDNAs from a 6-day-long, 2-day-interval experiment. The next time cDNA is made, the numbers could be switched to 002. The orange circles are stickers used to label the lid of the undiluted DNA. "1:10, 1:100, and 1:1000" designate the dilutions you will make.

cDNA labeling scheme


  • Transfer all 20 μL of cDNA sample 1 from the reaction tube into the first labeled tube (e.g., U2OS E001).
  • Add the following volumes molecular biology-grade H2O to the next three tubes
    • 1:10 = 90 μL
    • 1:100 = 45 μL
    • 1:1000 = 45 μL
  • Transfer 10 μL cDNA into the 90 μL H2O in the "1:10" tube. Mix thoroughly by flicking the tube.
  • Transfer 5 μL cDNA into the 45 μL H2O in the "1:100" tube. Mix thoroughly by flicking the tube.
  • Transfer 5 μL cDNA into the 45 μL H2O in the "1:1000" tube. Mix thoroughly by flicking the tube.
  • Repeat this process for all cDNA samples. There should be four tubes per cDNA sample: undiluted, 1:10 (for low-expressing genes), 1:100 (for intermediate-expressing genes), and 1:1000 (for highly-expressing genes like GAPDH, ACTB, and synthetic transgenes)
  • Store all cDNA at -20°C.

RNA Concentrations

Make sure to measure using 'single-stranded RNA' protocol on the Nanodrop instrument.

Cell Line Transfection Timepoint 260/280 ng/µL Volume (8 µL max) Mass (2 µg max)
MCF7KAH126-MV224 hr2.051693.4532.92.0
MCF7KAH126-MV224 hr2.046408.7674.92.0
MCF7no DNA24 hr2.04456.5894.42.0
MCF7KAH126-MV248 hr2.056346.2995.82.0
MCF7KAH126-MV248 hr2.046295.2146.82.0
MCF7no DNA48 hr2.051331.2116.02.0
MCF7KAH126-MV272 hr2.066687.9142.92.0
MCF7KAH126-MV272 hr2.052420.6974.82.0
MCF7no DNA72 hr2.06522.5813.82.0

oligo(dT) Primer-RNA annealing reactions

Sample ID Total RNA (µL) 50 µM oligo(dT) 10 mM dNTP mix Water (SS III kit)
MCF7 12.91.01.05.1
MCF7 24.91.01.03.1
MCF7 34.41.01.03.6
MCF7 45.81.01.02.2
MCF7 56.81.01.01.2
MCF7 66.01.01.02.0
MCF7 72.91.01.05.1
MCF7 84.81.01.03.2
MCF7 93.81.01.04.2

--> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.

cDNA synthesis mix

  • Total reactions = 9 (+1 buffer)
  • Samples:
  1. MCF7 1 (24 hr KAH126-MV2 rep1)
  2. MCF7 2 (24 hr KAH126-MV2 rep2)
  3. MCF7 3 (24 hr no DNA)
  4. MCF7 4 (48 hr KAH126-MV2 rep1)
  5. MCF7 5 (48 hr KAH126-MV2 rep2)
  6. MCF7 6 (48 hr no DNA)
  7. MCF7 7 (72 hr KAH126-MV2 rep1)
  8. MCF7 8 (72 hr KAH126-MV2 rep2)
  9. MCF7 9 (72 hr no DNA)
Reagent Single rxn. Mix (x10)
10x RT buffer 2.0 20.0
25 mM MgCl2 4.0 40.0
0.1 M DDT 2.0 20.0
RNaseOUT 1.0 10.0
SuperScript III RT 1.0 10.0
  10.0 μL 100.0 μL

--> Aliquot 10 μL of mix into 8-tube strip
--> Add annealing rxn. into each 10 μL aliquot
--> PCR machine: 50°C/ 50 min., 80°C/ 5 min., 4°C/ ∞
--> Add 1.0 μL RNase H, incubate at 37°C/ 20 min.
--> Store at -20°C


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