User:David Benjamin Nyer/Notebook/PcTF breast cancer/2016/03/28

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cDNA Synthesis from BT474 & BT549 RNA

I'll be measuring the concentration of the RNA samples from BT-474 & BT549 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.

oligo(dT) Primer-RNA annealing reactions

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

cDNA synthesis mix

  • Total reactions = 18 (+1 buffer)
  • Samples:
  1. BT474 1 (24hr no DNA)
  2. BT474 2 (24hr KAH126-MV2 rep A)
  3. BT474 3 (24hr (KAH126-MV2 rep B)
  4. BT474 4 (48hr no DNA)
  5. BT474 5 (48hr KAH126-MV2 rep A)
  6. BT474 6 (48hr (KAH126-MV2 rep B)
  7. BT474 7 (24hr no DNA)
  8. BT474 8 (24hr KAH126-MV2 rep A)
  9. BT474 9 (24hr (KAH126-MV2 rep B)
  10. BT549 1 (24hr no DNA)
  11. BT549 2 (24hr KAH126-MV2 rep A)
  12. BT549 3 (24hr (KAH126-MV2 rep B)
  13. BT549 4 (48hr no DNA)
  14. BT549 5 (48hr KAH126-MV2 rep A)
  15. BT549 6 (48hr (KAH126-MV2 rep B)
  16. BT549 7 (72hr no DNA)
  17. BT549 8 (72hr KAH126-MV2 rep A)
  18. BT549 9 (72hr (KAH126-MV2 rep B)


Reagent Single rxn. Mix (x19)
10x RT buffer 2.0 38.0
25 mM MgCl2 4.0 76.0
0.1 M DDT 2.0 38.0
RNaseOUT 1.0 19.0
SuperScript III RT 1.0 19.0
  10.0 μL 190.0 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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