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

PcTF breast cancer

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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 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. 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) MCF7 KAH126-MV2 24 hr 2.051 693.453 2.9 2.0 MCF7 KAH126-MV2 24 hr 2.046 408.767 4.9 2.0 MCF7 no DNA 24 hr 2.04 456.589 4.4 2.0 MCF7 KAH126-MV2 48 hr 2.056 346.299 5.8 2.0 MCF7 KAH126-MV2 48 hr 2.046 295.214 6.8 2.0 MCF7 no DNA 48 hr 2.051 331.211 6.0 2.0 MCF7 KAH126-MV2 72 hr 2.066 687.914 2.9 2.0 MCF7 KAH126-MV2 72 hr 2.052 420.697 4.8 2.0 MCF7 no DNA 72 hr 2.06 522.581 3.8 2.0 oligo(dT) Primer-RNA annealing reactions Sample ID Total RNA (µL) 50 µM oligo(dT) 10 mM dNTP mix Water (SS III kit) MCF7 1 2.9 1.0 1.0 5.1 MCF7 2 4.9 1.0 1.0 3.1 MCF7 3 4.4 1.0 1.0 3.6 MCF7 4 5.8 1.0 1.0 2.2 MCF7 5 6.8 1.0 1.0 1.2 MCF7 6 6.0 1.0 1.0 2.0 MCF7 7 2.9 1.0 1.0 5.1 MCF7 8 4.8 1.0 1.0 3.2 MCF7 9 3.8 1.0 1.0 4.2 --> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min. cDNA synthesis mix Total reactions = 9 (+1 buffer) Samples: MCF7 1 (24 hr KAH126-MV2 rep1) MCF7 2 (24 hr KAH126-MV2 rep2) MCF7 3 (24 hr no DNA) MCF7 4 (48 hr KAH126-MV2 rep1) MCF7 5 (48 hr KAH126-MV2 rep2) MCF7 6 (48 hr no DNA) MCF7 7 (72 hr KAH126-MV2 rep1) MCF7 8 (72 hr KAH126-MV2 rep2) 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