Hieff sybr green master mix

Total RNA from stable cells was extracted using the TRIzol reagent (15596026, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription procedure was described previously; three micrograms of the treated total RNA were used for cDNA synthesis in a 20 μL reaction using SuperScript II reverse transcriptase (18064022, Invitrogen, Carlsbad, CA, USA) [48 (link),49 (link),50 (link)].
qRT-PCR was performed using Hieff SYBR Green Master Mix (11201ES03, Yeasen Biotech, Shanghai, China), and reactions were performed on Roche LightCycler^® 480 II Real-Time PCR System. Data were acquired and analyzed using the 2^−ΔΔCT method with ACTIN as an endogenous control. Probes and primers used in qRT-PCR are listed in Table S1 (Supplementary Material).

Total RNA from the stored plants was extracted with TRIzol, and cDNA was obtained using the BeyoRT™ II cDNA kit (Beyotime, Shanghai, China). Quantitative analysis was performed by real-time PCR in conjunction with Hieff® SYBR Green Master Mix (Yeasen) on a LightCycler 480 System (Roche, Vienna, Austria) according to the manufacturer’s instructions. The primers used are listed in Table S15. The following reaction conditions were applied: predenaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and 65 °C for 60 s, and the melting curve was evaluated from 65 °C to 95 °C. The relative transcript levels of the candidate genes were calculated according to the 2^−ΔΔCT method. The melting peaks of the candidate genes are displayed in Fig. S3.

5 ml PB was collected on day 2, 7, 28 and 2, 3, 4, 5, 6, 9, and 12 months after CAR-T infusion to detect CAR-T count and copy number, which were used to assess expansion and persistence of CAR-T cells. FCM was used to evaluate the ratio of CAR-T cells to CD3⁺CD45⁺ T cells and the absolute number of CAR-T cells per μL of PB, namely, CAR-T count. Quantitative polymerase chain reaction (qPCR) for CD19 CAR, CD22 CAR, and total CAR-T gene copies was conducted on genomic DNA extracted from PB (TIANGEN, BEJ) using Hieff^™ SYBR^® GREEN Master Mix (Yeasen, SHH). CAR copies per μg DNA were normalized by the single-copy gene CDKN1a.