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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><br/><div class = "text-block">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.</div><br/><div class = "text-block">The original protocol is attached below:</div><div class = "text-block"><span>Kit:  </span><a href="https://www.thermofisher.com/order/catalog/product/18080051#/18080051" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Script™ III First-Strand Synthesis System, ThermoFisher Catalog number: 18080051</span></a><span></span></div></div>

[STEPS]
?. 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.
?. Label two PCR tubes per sample, RT and NRT.
Best practice for qPCR is to prepare a no reverse transcriptase (NRT) reaction for each of your samples to ensure that minimal genomic DNA remains in your reverse transcriptase sample and that only cDNA, not gDNA, is being interpreted as gene expression in your qPCR results.
?. 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  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.ComponentAmount for 1 rxnAmount for 10 rxn50 ng/ul Random Hexamers1 ul10 ul10 mM dNTP Mix1 ul10 ulDEPC-treated or MilliQ (18.2 MΩ.cm) water3 ul30 ul
5 µl
ComponentAmount for 1 rxnAmount for 10 rxn50 ng/ul Random Hexamers1 ul10 ul10 mM dNTP Mix1 ul10 ulDEPC-treated or MilliQ (18.2 MΩ.cm) water3 ul30 ul
?. Add  of high quality RNA from each sample to the respective RT and NRT tubes.
5 µl
Keep RNA samples on dry ice when not actively in use to inhibit RNases.  Return samples to   as soon as possible.
?. Incubate the tube at  for   in the thermocycler.
65 °C
?. Place  for a minimum of .
on ice
?. 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  when not in use.
on ice
?. Prepare a cDNA Synthesis (RT) Master Mix adding each component in the indicated order. Adjust the table for the number of RT samples.OrderComponentAmount for 1 rxnAmount for 10 rxn110X RT buffer2 ul20 ul225 mM MgCl24 ul40 ul30.1 M DTT2 ul20 ul440 U/ul RNase OUT1 ul10 ul5200 U/ul SuperScript III RT1 ul10 ul
OrderComponentAmount for 1 rxnAmount for 10 rxn110X RT buffer2 ul20 ul225 mM MgCl24 ul40 ul30.1 M DTT2 ul20 ul440 U/ul RNase OUT1 ul10 ul5200 U/ul SuperScript III RT1 ul10 ul
?. Add  of the cDNA Synthesis (RT) Master Mix to each tube labeled RT.
10 µl
?. Prepare a cDNA Synthesis (NRT) Master Mix adding each component in the indicated order. Adjust the table for the number of NRT samples.OrderComponentAmount for 1 rxnAmount for 10 rxn110X RT buffer2 ul20 ul225 mM MgCl24 ul40 ul30.1 M DTT2 ul20 ul440 U/ul RNase OUT1 ul10 ul5DEPC-treated or MilliQ (18.2 MΩ.cm) water1 ul10 ul
OrderComponentAmount for 1 rxnAmount for 10 rxn110X RT buffer2 ul20 ul225 mM MgCl24 ul40 ul30.1 M DTT2 ul20 ul440 U/ul RNase OUT1 ul10 ul5DEPC-treated or MilliQ (18.2 MΩ.cm) water1 ul10 ul
?. Add of the cDNA Synthesis (NRT) Master Mix to each tube labeled NRT.
10 µl
?. Program a thermocycler for the following and run all samples, both RT and NRT:StepTime (m:s)Temp (C)110:0025250:005035:00854Infinity4The samples can be removed after the 5 minute incubation at 85oC is complete, they do not need to reach 4oC in the thermocycler.
StepTime (m:s)Temp (C)110:0025250:005035:00854Infinity4
?. Chill  until samples are cool to the touch.
on ice
?. Treat gloves and pipettes with RNase away  and sterilize work area with 70% ethanol before pipetting reagents.
?. Briefly minicentrifuge all samples.
?. Add  of RNase H to each tube (both RT and NRT) and incubate in a thermocycler for  at
1 µl
37 °C
?. cDNA samples can be stored at .
-20 °C