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

Material

Input sample requirements

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.
Sample type Starting material per 1 mL of TRIzolTM Reagent
Tissues[1] 50 ー 100 mg of tissue
Cells grown in monolayer 1 × 10xx5 ー 1 × 10xx7 cells grown in monolayer in a 3.5–cm culture dish (10 cm2)
Cells grown in suspension 5 ー 10 × 10xx6 cells from animal, plant, or yeasty origin or 1 × 10xx7 cells of bacterial origin
[1] Fresh tissues or tissues stored in RNAlaterTM Stabilization Solution (Cat. No. AM7020).

Procedural guidelines

  • 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.



Protocol

1 Lyse samples and separate phases
  • 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.
2 RNA binding and DNaseI treatment
  • 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).










Material

  • 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)

Components Volume Storage Memo
PrimeScript II RTase(200 U/μl) 50 μl -20°C
5×PrimeScript II Buffer 200 μl -20°C
RNase Inhibitor(40 U/μl) 25 μl -20°C
dNTP Mixture (10 mM each) 50 μl -20°C
Oligo dT Primer(50 μM) 50 μl -20°C
Random 6 mers(50 μM) 100 μl -20°C
RNase free dH2O 1 ml -20°C




Protocol

1 Reaction preparation
  • Mix the following reagents in one tube
Reagent Volume for 1 reaction (µL) Volume for 10 reactions (µL) Note
Oligo dT Primer(50 μM) 1 10
dNTP Mixture(10 mM each) 1 10
RNA Template ~8 50 Total RNA:<5 μg, polyA+ RNA:<1 μg
RNase Free dH2O up to 10 30
Total 10 100
  • 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.



2 Reverse transcription
  • Prepare master mix for reaction;
Reagent Volume per 1 reaction (µL) Volume per 5 reaction (µL) Note
5×PrimeScript II Buffer 4 20
RNase Inhibitor(40 U/μl) 0.5 2 20U per reaction
PrimeScript II RTase(200 U/μl) 1 5 200U per reaction
RNase Free dH2O 4.5 22.5
  • 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
Temprature Time Note
45˚C 60 min
75˚C 15 min Deactivation. Paramater for long transcrpits just in case.
4˚C Forever (Immediately put on ice.)


  • 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.







Protocol last updated
  • interactome/experiments/20200329.1585485400.txt.gz
  • Last modified: 2020/03/29 21:36
  • by dan