Haynes:cDNA Synthesis

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SuperScript III

by Karmella Haynes, 2014

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

A. 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 MgCl_{2, 0.1 M DDT}
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 fromt he 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)

B. 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.

Your NOTEBOOK ENTRIES should contain the following tables and information. Switch to edit mode on this page to copy-paste the code for the tables:

Measure RNA concentration

Sample	OD 260	260/280	ng/μL	Vol. to use for cDNA synth.
1. ###	###	###	###	### μL (### μg)
2. ###	###	###	###	### μL (### μg)

oligo(dT) Primer-RNA annealing reactions

Reagent	Vol
total RNA (up to 2μg)	up to 8 μL
50 μM oligo(dT) primer	1.0
10 mM dNTP mix	1.0
Water (SS III kit)	= 8.0 - vol. total RNA
	10.0 μL

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

cDNA synthesis mix

• Total reactions = N

• Samples:

1. Names & description of cDNA sample 1
2. Names & description of cDNA sample 2
3. Names & description of cDNA sample 3
4. etc.

Reagent	Single rxn.	Mix (xN)
10x RT buffer	2.0	###
25 mM MgCl2	4.0	###
0.1 M DDT	2.0	###
RNaseOUT	1.0	###
SuperScript III RT	1.0	###
	10.0 μL	### 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