Abi prism 7500

RNA was reverse transcribed into cDNA from 8 ng (frozen tissue) and 50 ng (blood) of total RNA using random primers and SuperScript III First-Strand Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions.
The qPCR was performed on ABI PRISM 7500 using SsoAdvanced™ Universal SYBR^® Green SuperMix (Bio-Rad, Hercules, CA, USA) with specific primers (listed in Supplementary Table S5). Reactions consisted of 1 µL of cDNA, 500 nM of each forward and reverse primer, 5 µL of qPCR Master Mix and 3 µL of nuclease-free water in thermal cycling conditions provided by the manufacturer. All reactions were performed in triplicates and ß-actin was used as an endogenous control. RT-qPCR data were analyzed using the comparative Ct Method [56 (link)].

PSC samples were collected to determine the effects of RNAi on the expression of EmTUBB3 on days 3, 6, 10 and 15. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After treatment with DNase I (Thermo, Waltham, MA, USA) for 30 min at 37 °C to remove genomic DNA contamination, the RNA samples were transcribed into cDNA using a reverse transcription kit (TaKaRa Bio, Dalian, China). qRT–PCR was performed using 2 μL of 1:5 diluted cDNA and SYBR Green Real-time PCR Master Mix (TaKaRa Bio, Dalian, China) and run in a thermocycler (ABI Prism 7500, Bio-Rad, Hercules, CA, USA). The following specific primers for EmTUBB3 and EF-1α (internal control) were synthesized by Sangon Biotech (Shanghai, China): EmTUBB3, 5′-AGGCTTGCGACTGCTTG-3′ (forward), 5′-CCGTGTCCGACACCTTAGGT-3′ (reverse); EF-1α, 5′-TTTGAGAAAGAGGCGGCTGAGATG-3′ (forward), 5′-TAATAAAGTCACGATGACCGGGCG-3′ (reverse). The qRT–PCR protocol was comprised of 39 cycles at 95 °C for 5 s, 60 °C for 10 s and 72 °C for 30 s. Each reaction was run in triplicate, after which the average threshold cycle (Ct) was calculated per sample and the relative expression of the genes was calculated using the 2^−ΔΔCT method [32 (link)].

Quantitative real-time PCR (qPCR) was performed to confirm the transcriptome results. Total RNA (1.5 μg) was reverse transcribed to cDNA with the SMARTerTM PCR cDNA Synthesis Kit (Clontech Laboratories Inc, USA), as per the manufacturer's instructions, and qPCR was carried out using an ABI Prism^® 7500 (Bio-Rad, USA) with SYBR Premix Ex TaqII (TAKARA, Japan) by following the instructions of the operation manual. Each reaction was replicated three times per biological replicate, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as the internal reference gene. The relative quantification of the transcript levels was performed using the 2^−ΔΔCT method. The 20 μl reaction volumes contained 2 μl cDNA, 0.4 μl L/R primers (10 μM), 10 μl 2 × qPCR SYBR Premix Ex TaqII, and 7.2 μl double-distilled water. Sequences of the primers for qPCR used in this study are listed in Table 1.