What are the most critical steps in the QuantSeq library generation ?

The most critical steps of QuantSeq FWD library generation are during first strand cDNA synthesis. Our detailed handling guidelines ensure the sample temperature is maintained at the optimal range of 42 degrees Celcius. This ensures specific hybridization of the oligo(dT) primer at transcript poly(A) tails and prevents internal priming (mishybridization).

For first time QuantSeq users, we recommend watching our QuantSeq's First Strand Synthesis instructional video.

First Strand cDNA Synthesis Tips - High Quality RNA Input

1. Prepare your RNA in 5 μl volumes and bring to room temperature for 2-5 minutes before adding the FS1 buffer.

2. Ensure FS1 is also fully equilibrated to room temperature. ATTENTION! Do not place RNA / FS1 samples back on ice and proceed immediately to denaturing after FS1 is added.

3. At step 3, pre-warm the FS2 / E1 mastermix for 2 – 3 minutes at 42 °C while the RNA / FS1 samples are denaturing for 3 minutes at 85 °C – Do not cool the mastermix on ice!

4. After the RNA / FS1 samples have cooled to 42 °C, spin these down briefly and then immediately return to the thermocycler and hold at 42 °C.

5. Add the pre-warmed FS2 / E1 mastermix to the RNA / FS1 samples on the thermocycler at 42 °C (step 4) and mix properly. Any drop in temperature at this point can cause mishybridization! Seal the plate or tubes and begin the 42 °C incubation.

6. After the 42 °C incubation is complete, proceed immediately to RNA Removal. Do not cool the samples on ice, or store below room temperature at this point!
   

NOTE: Spin down the samples at room temperature before and after adding the FS2 / E1 mastermix.

First Strand cDNA Synthesis Handling Tips - Low input, degraded and FFPE samples

1. Prepare your RNA samples in 5 μl volumes.

2. Prepare a mastermix containing 5 μl FS1, 9.5 μl FS2, and 0.5 μl E1, mix well, spin down, and pre-warm at 42 °C on a thermocycler for 2 – 3 minutes.

3. Bring your RNA samples to room temperature for 2 – 5 minutes while the mastermix is pre-warming. Adding mastermix to cooled RNA samples can result in mishybridization!

4. Spin down the pre-warmed FS1 / FS2 / E1 mastermix and add 15 μl to each RNA sample. Quickly mix, seal the plate or strip-tubes, spin down briefly at room temperature, and then commence the 42 °C incubation for 15 minutes on a thermocycler (or 1 hour for low input RNA (≤ 10 ng)).

5. After the 42 °C incubation is complete, proceed immediately to RNA Removal. Do not cool the samples on ice, or store below room temperature at this point!
   

RNA Removal Handling Tips

Proceed immediately to the RNA removal after the reverse transcription is complete! (step 4 – 5). Do not place the samples on ice, and do not store samples at -20 °C at this point! Cooling the samples below room temperature at this point can cause mishybridization! Best practice handling would be as follows:

1. After the 42 °C incubation is complete, spin down the plate/tubes briefly and place at room temperature (or place back on the thermocycler at 42 °C)

2. Remove the sealing foil / tube caps, and immediately add the RNA Removal Solution (RS, thawed at room temperature) to the samples, mix well.

3. Briefly spin down the plate / tubes at room temperature, then place on the thermocycler to commence the 10 minute incubation at 95 °C (step 6).

NOTE: To minimize temperature drops at this point, the reactions can also be kept at 42 °C while the RNA Removal Solution (RS) is added.