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.
- 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
- 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.
- 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).
- Retrieve the SuperScript III kit from the -20°C freezer.
- 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
- Keep the following on ice or in a cold block: SuperScript III RT, RNaseOUT, RNase H.
- NOTEBOOK ENTRY: In labeled, clean RNase-free 0.5 mL tubes, set up oligo(dT) Primer-RNA annealing reactions.
- Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.
- 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).
- Transfer each primer-RNA annealing reaction into a 10 μL aliquot of cDNA synthesis mix.
- 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/ ∞
- 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.
- 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.
- 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.
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)
- BT474 1 (24hr no DNA)
- BT474 2 (24hr KAH126-MV2 rep A)
- BT474 3 (24hr (KAH126-MV2 rep B)
- BT474 4 (48hr no DNA)
- BT474 5 (48hr KAH126-MV2 rep A)
- BT474 6 (48hr (KAH126-MV2 rep B)
- BT474 7 (24hr no DNA)
- BT474 8 (24hr KAH126-MV2 rep A)
- BT474 9 (24hr (KAH126-MV2 rep B)
- BT549 1 (24hr no DNA)
- BT549 2 (24hr KAH126-MV2 rep A)
- BT549 3 (24hr (KAH126-MV2 rep B)
- BT549 4 (48hr no DNA)
- BT549 5 (48hr KAH126-MV2 rep A)
- BT549 6 (48hr (KAH126-MV2 rep B)
- BT549 7 (72hr no DNA)
- BT549 8 (72hr KAH126-MV2 rep A)
- BT549 9 (72hr (KAH126-MV2 rep B)
|| Single rxn.
|| Mix (x19)
| 10x RT buffer
| 25 mM MgCl2
| 0.1 M DDT
| SuperScript III RT
|| 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