Real-time reverse transcription PCR (for Dual pH-sensitive nanodrug blocks PD-1 immune checkpoint and uses T cells to deliver NF-κB inhibitor for antitumor immunotherapy)

1. Total RNA extraction

1) Cells were washed twice with ice-cold PBS;

2) Add 1 mL of TRIzol to each well of a six-well plate, incubated for 10 min, and transferred to a 1.5 mL RNAase-free centrifuge tube;

3) Add 200 μL of chloroform per 1 mL TRIzol, gently vortexed for 15 sec, let the mix sit for 5 min, and then centrifuged at 12500 rpm for 15 min at 4 ℃;

4) Collected the upper layer of clear liquid, equal volume of isopropanol was added, gently vortexed for 15 sec, let the mix sit for 10 min, and centrifuged at 12500 rpm for 10 min at 4 ℃.

5) The RNA was pelleted by centrifugation at 8000 rpm, washed with cold 75% ethanol, and dissolved in an appropriate volume of RNase-free water.

2. RNA concentration detection and Reverse transcription (RT) reaction.

1) The quantity and purity of RNA were measured using NanoDrop (NanoDrop Technologies, USA);

2) Reverse transcription reaction was conducted with the reverse transcription kit (TaKaRa, China):

a. RT reaction liquid:

Reagent	Usage	Final concentration
5×PrimeScript RT Master Mix（Perfect Real Time）	2 μL	1×
Total RNA	According to the RNA concentration
RNase Free dH₂O	Up to 10 μL

*Note: The reaction mixture (10 μL) for cDNA synthesis consisted of 2 μL RT Master Mix, 0.5 μg total RNA, and RNase Free dH₂O.

b. The conditions of RT reaction:

Temperature	Reaction time
37℃	15 min	Reverse transcription reaction
85℃	5 sec	Transcriptase inactivation
4℃	∞

3. Quantitative Real-time PCR

Quantitative Real-time PCR was performed on an ABI Real-Time PCR System (Applied Biosystems, USA).

a. qRT-PCR reaction included 10 μL SYBR Green q-PCR SuperMix (Roche, USA), 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 1 μL cDNA, and 9 μL ddH₂O.

b. PCR program: 95 ℃ 10 min; 40 repeats of 95 ℃ 10 sec, 60 ℃ 45 sec, 95 ℃ 15 sec; 60 ℃ 1 min, 95 ℃ 15 sec.

4. Quantitative Real-time PCR data

The relative value of mRNA expression was calculated by the comparative ΔΔC_t method using β-actin as a reference gene.