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===First strand cDNA Synthesis=== :- Thaw all the reagents needed for first-strand cDNA synthesis (except the enzyme) on ice. Gently vortex each reagent to mix and spin down briefly. Store on ice. :- Prepare a stock solution of 10X Reaction Buffer by mixing the 10X Lysis Buffer - v3 with the RNase Inhibitor as indicated below (scale-up as needed), Vortex briefly to mix, then spin down: :{| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''volume''' | align="center" style="background:#f0f0f0;"|'''component''' |- | 19 μl||10X Lysis Buffer v3 |- | 1 μl||Rnase Inhibitor |- | 20 μl||Total Volume |} :- Prepare each sample (10 μl total volume) in individual 0.2 ml RNase-free PCR tubes or in an 8-well strip. :{| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample A''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample B''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample C''' |- | 10X Reaction Buffer||0.5 ul ||0.5 ul ||0.5 ul |- | Nuclease-free water||3.5 ul ||3.5 ul ||3.5 ul |- | Diluted Control RNA||1 ul ||1 ul ||1 ul |- | Total volume||5 ul || 5 ul || 5 ul |} :- Place the samples on ice, and add 3’ SMART CDS Primer II A (12 μM) like below. Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube: :{| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''volume''' | align="center" style="background:#f0f0f0;"|'''component''' |- |5 μl||Cell/Total RNA in Reaction Buffer |- |0.5 μl||3’ SMART CDS Primer II A (12 μM) |- |5.5 μl||Total Volume |} :- Place the tubes into a pre-heated thermal cycler and run 72°C for 3 min, then quickly put them on ice. :- Pre-heat the thermal cycler to 42°C again :- Meanwhile, prepare enough Master Mix for all the reactions, plus 10% of the total reaction mix volume, by combining the following reagents in the order shown at room temperature: :{| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''volume per reaction''' | align="center" style="background:#f0f0f0;"|'''volume for 3 reactions''' | align="center" style="background:#f0f0f0;"|'''component''' |- | 2 μl|| 6ul || 5X First-Strand Buffer |- | 0.25 μl||0.75 ul || DTT (100 mM) |- | 0.5 μl||1.5 ul || dNTP Mix (20 mM) |- | 1 μl||3 ul || SMARTer IIA Oligonucleotide (12 μM) |- | 0.25 μl||0.75 ul ||RNase Inhibitor (40 U/μl) |- | 1 μl||3ul ||SMARTScribe Reverse Transcriptase (100 U/μl)* |- | 5 μl|| 15 ul|| Total Volume added per reaction |} :- Add the reverse transcriptase to the Master Mix just prior to use, making sure to gently mix the reverse transcriptase tube without vortexing before adding it. :- Mix the Master Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube. :- Place the tubes in a thermal cycler pre-heated to 42°C. Run the following program: :- 42°C 90 min -> 70°C 10 min -> 15°C forever :- STOPPING POINT: The tubes can be stored at 4°C overnight.
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