TITLE: cDNA Synthesis Using SuperScript III First-Strand Synthesis System for RT-PCR
AUTHORS: Lynn    Doran

[DESCRIPTION]
The SuperScript® III First-Strand Synthesis System for RT-PCR is used to synthesize first-strand cDNA from purified  total RNA. RNA targets from 100 bp to >12 kb can be detected with this system. 

The procedure follows the manufacturer's instructions but includes lab specific template and reagent amounts and specifies primers and equipment for use in our lab to prepare total RNA samples for qPCR analysis of gene expression.


The original protocol is attached below:
  

Kit:  Script™ III First-Strand Synthesis System, ThermoFisher Catalog number: 18080051

[BEFORE_START]
RNA extracted from Qiagen RNeasy Plant Extraction Kit, or equivalent quantity and quality.
RNA used for cDNA synthesis for qPCR should be high quality.
A260/A280 values  > 1.8, ideally values approach 2.1 (note in SYBR green guide they recommend using samples with >2).
There is no defined rule for an acceptable A260/A230 ratio for qPCR.  A low value is often the result of guanidium thiocyanate contamination from the extraction procedure, and that concentrations of up to 100 mM are tolerated. A good rule of thumb, A260/A230 values should be > 1.8, and for pure RNA expect values 2.1-2.3.
RNA integrity as indicated by Qubit RNA IQ values >5, ideally > 8. 

[GUIDELINES]
Always wear gloves  and change them often to avoid RNases.
Use RNase-free solutions. 
Use RNase-free certified, disposable plasticware and filter tips whenever possible.

[STEPS]
1. Allow reagents to thaw completely, mix, and briefly minicentrifuge 10 mM dNTP mix and 50 ng/ul random hexamers before use. Store on ice when not in use.
2. Label two PCR tubes per sample, RT and NRT.
 

3. Treat gloves and pipettes with RNase away  and sterilize work area with 70% ethanol before pipetting reagents.  
4. If performing many reactions, make a master mix of primer random hexamers, dNTP mix, and water, multiply each component by the number of reactions needed plus at least 20% to account for pipetting error. Remember that you will need two reactions for each sample.  Pipette 5 µL of the prepared master mix into each sample tube, both RT and NRT.

 If only a few reactions are needed, pipette the following for 1 reaction into each tube.

 

5. Add 5 µL of high quality RNA from each sample to the respective RT and NRT tubes.
 


6. Incubate the tube at 65 °C for 5 min  in the thermocycler.

7. Place on ice for a minimum of 1 min.

8. Allow reagents to thaw completely, mix,  and briefly centrifuge 10X RT buffer, 25 mM MgCl2, 0.1 M DTT, 40 U/ul RNase OUT, and 200 U/ul SuperScript III RT before use. Store on ice when not in use.

9. Prepare a cDNA Synthesis (RT) Master Mix adding each component in the indicated order. Adjust the table for the number of RT samples.


 

10. Add 10 µL of the cDNA Synthesis (RT) Master Mix to each tube labeled RT.

11. Prepare a cDNA Synthesis (NRT) Master Mix adding each component in the indicated order. Adjust the table for the number of NRT samples.
 
 

 
12. Add 10 µLof the cDNA Synthesis (NRT) Master Mix to each tube labeled NRT.

15. Program a thermocycler for the following and run all samples, both RT and NRT:

 

The samples can be removed after the 5 minute incubation at 85oC is complete, they do not need to reach 4oC in the thermocycler.
16. Chill on ice until samples are cool to the touch.

17. Briefly minicentrifuge all samples.

18. Add 1 µL of RNase H to each tube (both RT and NRT) and incubate in a thermocycler for 20 min at 37 °C

19. cDNA samples can be stored at -20 °C.

13. Incubate at 665 °Cfor 5 min.
14. Place on ice for at least 1 minute.