2.4. RNA Extraction From PBMCs, cDNA Reverse Transcription, and Quantification Through Real‐Time qPCR

1- RNA recovery from RNAzol/TRIzol-homogenized cells

Work on ice otherwise differently stated!

1. Add 200 μl RNAzol/1^E6 to the cell pellet and vigorously pipet to homogenize.

1. Freeze at -80°C or proceed to RNA extraction step 3. 

1. Add 1:5 chloroform per sample.

1. Shake vigorously/invert tubes for 15 seconds at RT to mix well.

1. Incubate for 15 min at 4°C.

1. Centrifuge for 15 min at 4°C 12000 rpm.

1. After centrifugation, a three-phase gradient will be visible:

• Transparent/aqueous upper phase → RNA (RNA remains in suspension)
• Thin white interphase → DNA 
• Pink lower phase → proteins + waste/debris

8. Transfer the upper aqueous (transparent) phase containing RNA into a new 1.5 mL microtube. Pay attention not to transfer any interphase (white, DNA) or phenol (pink). For 200 μl RNAzol you should recover nearly 100 μl of aqueous phase. 

9. Add 1:1 isopropanol to the recovered aqueous phase.

10. Mix by inverting the tubes up and down continuously for 2 min. 

11. Incubate for 1.5h at -80° or overnight at -20°C (best option) to encourage RNA precipitation.

12. The day after, incubate samples for 10 min at 4°.

13. Centrifuge for 10 min at 4°C 12000 rpm.

14. Discard supernatant and wash RNA pellet by adding 100 μl of chilled 75% ethanol (3 times).

15. Centrifuge 10 min at 4°C 12000 rpm after each wash.

16. Thoroughly remove residual ethanol from the last wash in two stages: 

-First with a pipette set at 100 μL 

-Then with a pipette set at 10 μL to clear the RNA pellet.

17. Let the pellet dry for 5-10 min under the laminar flow hood at RT by leaving caps open. It should become almost transparent but not excessively dried. 

18. Resuspend pellet in 11 μl of nuclease-free/molecular grade H₂O.

19. Use 1 μl for RNA quantification at NanoDrop. Remember to clean the NanoDrop thoroughly and perform blank measurement first.

RNA Quality Ratios 

A260/A280 and A260/A230 ratios > 1.8 indicate acceptable RNA purity

20 Store eluted RNA at -80° or continue with reverse transcription.

2-Reverse Transcription

Work on ice otherwise differently stated!

1.Pipette the volume of RNA required to obtain 2 μg of total RNA and adjust the final volume to 14 μL using nuclease-free (molecular biology grade) water.

 SuperScript VILO™ cDNA Synthesis Kit (Invitrogen by Thermo Fisher Scientific)

 2. Add 4 μL of 5X VILO™ Reaction Mix and 2 μL of 10X SuperScript Enzyme Mix, arriving at a final volume of 20 μL.

3. Mix thoroughly and spin.

 4. Place the samples in a thermal cycler and run the following program:

• Primer annealing         25°C    10 min
• Reverse transcription              42°C    60 min
• Enzyme inactivation  85°C    5 min
• Hold      4°C      ∞

5. Upon completion of reverse transcription, you can either freeze, or dilute samples 1:8 by adding 140 μL H₂O (this should yield a 25ng equivalent of cDNA for 2 μl volume to be used in the qPCR reaction).

6. Place cDNA at 4°C/on ice for immediate use, at −20°C for routine use, or at −80°C for long-term storage.

3- Real-Time PCR (qPCR)

Keep all reagents and enzymes on ice and prepare the PCR reaction!

Before loading cDNA on the wells, vortex each sample thoroughly!

1. Prepare the reaction mix (final volume 10 ul), as follows for and dispense in each well:

• 5 μL of SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) 
• 0.5 μL of already customized primers (PrimePCR SYBR Green Assay, Bio-Rad) 
• 2.5 μL H₂O

and add

• 2 μL of cDNA to each well

2. Seal the plate with optical adhesive film and press firmly with a spatula or roller to prevent edge leakage.

3. Briefly centrifuge the PCR plate and load it into the thermocycler (CFX Manager Software, BioRad) according to the following thermal profile:

x 40 cycles 

• initial denaturation 95°C at 15 min 
• denaturation 15 s at 95°C 
• annealing 1 min at 60°C 
• extension 20 s at 72°C