Based on the user guide of Trizol reagent by Invitrogen and Column kit (ZYMO).

IMPORTANT!
Perform RNA isolation immediately after sample collection or quick-freeze samples immediately after collection and store at –80°C or in liquid nitrogen until RNA isolation.

• Perform all steps at room temperature (20–25°C) unless otherwise noted.
• Use cold TRIzolTM Reagent if the starting material contains high levels of RNase, such as spleen or pancreas samples.
• Use disposable, individually wrapped, sterile plastic ware and sterile, disposable RNase-free pipettes, pipette tips, and tubes.
• Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin; change gloves frequently, particularly as the protocol progresses from crude extracts to more purified materials.
• Always use proper microbiological aseptic techniques when working with RNA.
• Use RNaseZapTM RNase Decontamination Solution (Cat.no. AM9780) to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.

• Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.

Cell grown in monolayer:

• Remove growth media.
• Add 0.3–0.4 mL of TRIzolTM Reagent per 1 × 105—107 cells directly to the culture dish to lyse the cells.
• Pipet the lysate up and down several times to homogenize.

Note: The sample volume should not exceed 10% of the volume of TRIzolTM Reagent used for lysis.

STOPPING POINT
Samples can be stored at 4°C overnight or at – 20°C for up to a year.

• (Optional) If samples have a high fat content, centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear supernatant to a new tube.
• Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex.
• Add 0.2 mL of chloroform per 1 mL of TRIzolTM Reagent used for lysis, then securely cap the tube.
• Incubate for 2–3 minutes.
• Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.
• The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase.
• Transfer the aqueous phase containing the RNA to a new tube. Transfer the aqueous phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.

• Add same volume of EtOH to the aqueous phase from the previous step.
• Transfer tube to the Zymo spin IICR Column in a collection tube and centrifuge for 30 seconds (@12,000 xg).
• Discard flow-through
• Prewash the column with 400 µL of RNA Wash Buffer and centrifuge for 30 seconds (@12,000 xg).
  *Discard flow-through
• To each reaction, add a Master mix containing 5 µL of DNaseI and 75 µL 1 x DNaseI buffer.
• Incubate at room temperature for 15 minutes.
• Add 700 µL of RNA Wash Buffer to the column and and centrifuge for 30 seconds (@12,000 xg).
  *Add 400 µL of RNA Wash Buffer and centrifuge for 2 min (@12,000 xg) to remove all remaining buffer.
  *Transfer column carefully to a new RNase-free tube.
• Add 50 µL of ddH2O to the column and centrifuge for 30 seconds (@12,000 xg).
  

• Sample : Total RNA sample from 20191122 (HEK293T RNA -1)
• PrimeScript™ II 1st strand cDNA Synthesis Kit (TaKara, Cat.6210A)

PrimeScript™ II 1st strand cDNA Synthesis Kit (For 50 reactions)

• Mix the following reagents in one tube

• Aliquot to 10 x 10 µL
• Incubate at 65˚C for 5 mins
• Transfer tube to ice immediately

! IMPORTANT STEP ! By doing this, the RNA template degenerates and the reverse transcription efficiency increases.

• Prepare master mix for reaction;

• Add 10 µL of the master mix to each tube to make the total volume 20 µL.

• Mix gently

• Immediately proceed to the following reaction

• Dilute reaction to 50 μl with 30 μl H2O for PCR.
• The cDNA product should be stored at -20°C.
• For downsteam PCR amplification, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.