Primescript reagent kit with gdna eraser

Single-stranded cDNA was synthesized using 2 μg of total RNA from each sample using a PrimeScript^TM reagent Kit with gDNA Eraser (TaKaRa, Dalian, Liaoning, China), following the instruction in the manual. Gene-specific primers were designed using Primer Premier 5.0 software (Table S3). RT-PCR programs were 98 °C for 2 min, followed by 25–32 cycles of 98 °C for 15 s, 52–58 °C for 10 s, and 72 °C for 1 min. A total volume of 10 μL PCR reaction system was performed containing 8.4 μL of Golden Star T6 Super PCR Mix (TsingKe, Beijing, China), 0.3 μL of forward primer (10 mM), 0.3 μL of reverse primer (10 mM), and 1.0 μL of cDNA templates (200 ng/μL). Amplification products were analyzed on 1.0% agarose gels. RpL3 (ribosomal protein L3) was used as an endogenous control gene to determine the consistency of the cDNA template concentration.

Total RNA was extracted from the infiltrated leaves using Trizol reagent (Invitrogen, Carlsbad, CA, United States) and 1 mg of RNA was retrotranscribed into cDNA using the PrimeScript^TM reagent kit with gDNA eraser (TaKaRa, Dalian, Japan) according to the manufacturer’s protocol. The RT-qPCR was performed in triplicates using a Roche Light Cycler 96 system (Roche Applied Science, Basel, Switzerland) with the following program: 30 s at 95°C, 45 cycles of 5 s at 95°C, 30 s at 58°C, and 10 s at 72°C. The specificity of primer pairs was verified by RT-qPCR dissociation curve. The relative expression level was calculated using the comparative Cq (2^–ΔΔCq) method. NbActin was used as an internal standard. The information of the primers used in the RT-qPCR experiments is listed in Supplementary Table S1.

Total RNA was extracted from the cells using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Single-strand cDNA was synthesized from 1 μg of total RNA using the Prime-ScriptTM Reagent Kit with gDNA Eraser (Takara, Dalian, China). Real-time quantitative PCR (RT-qPCR) primers were purchased from the Beijing Genomics Institute. The primers were as follows: PLAU sense, 5′-TCACCACCAAAATGCTGTGT-3′, and antisense, 5′-CCAGCTCACAATTCCAGTCA-3′ (Xu et al., 2015 (link)). The qPCR was conducted on a 7300 Real-Time PCR system (Applied Biosystems, Life Technologies, Singapore). The following cycling conditions were applied: 95 °C for 5 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 30 s. For each sample, qPCR assays were conducted in triplicate in a 10 μl reaction volume. β-Actin served as an internal control to normalize the expression of PLAU. The 2^−ΔΔCt method was employed to calculate the relative expression of PLAU mRNA (Livak & Schmittgen, 2001 (link)).

The total RNA was extracted from tissue with reagent (Life Technologies, Carlsbad, California) and reverse-transcribed to cDNA by Prime Script TM Reagent Kit with gDNA Eraser (Takara Bio, Shiga, Japan). Quantitative real-time (QRT) PCR was performed using cDNA as template by the QRT-PCR detection systems (ABI7500, Applied Biosystems, United States). The qPCR reaction system conditions were 95°C 30 s; 95°C 5 s, 60°C 10 s, and 72°C 15 s, with a total of 40 cycles. Using GAPDH as internal reference, the relative expression of mRNA of target genes was calculated by the 2^−ΔΔCt method.

Total RNA was extracted from 15 WT, shank3a^–/–, shank3b^–/–, and shank3ab^–/– larvae each at 3.5–4.5 months post-fertilization (mpf) using the RNA Extraction Kit from Takara, and reverse transcribed to cDNA using the PrimeScript^TM reagent Kit with gDNA Eraser (Takara) according to the manufacturer’s recommendations. The Cas9 target region of shank3a and shank3b were amplified in duplicate samples from shank3ab^–/– zebrafish by real-time quantitative PCR (RT-qPCR) to confirm genotype (Figures 1C,D and Supplementary Table 1).
To assess the effect of VPA exposure on synaptic genes and class I hdac genes (as VPA belongs to class I HDAC inhibitor), groups of ∼15 WT and shank3ab^–/– zebrafish larvae were exposed to vehicle or VPA from 4 to 8 dpf, reared under normal conditions, then sacrificed for whole-brain total RNA isolation. The expression levels of the following genes analyzed by RT-qPCR: NMDAR subunits (grin1a, grin1b, grin2bb, grin2ca, grin2da, grin2aa), AMPAR subunits (gria1a, gria1b, gria2b), mGluR subunits (grm1a, grm1b, grm5a), and class I hdacs (hdac1, hdac3, hdac8). We selected β-actin or Rpl13α as internal controls because both are expressed in the brain throughout development. Primer sequence are shown in Supplementary Table 1.

TRIzol reagent (Invitrogen, USA) was used to extract total RNA in accordance with manufacturer’s instructions. MiR-150-5p expression was detected by a Hairpin-it^TM microRNA and U6 snRNA normalization RT-PCR quantitation kit (Genepharma, China). For VEGFA mRNA, complementary DNA (cDNA) was synthesized using PrimeScript^TM reagent kit with gDNA Eraser (Takara, Dalian, China) and qRT-PCR was analyzed using SYBR Premix Ex Taq kit (Takara, Dalian, China). The relative expression of miRNA or mRNA was analyzed using the 2^-ΔΔCT method. All results were normalized to GAPDH or U6 gene. The primers for VEGFA and GAPDH mRNA are listed below (Table 3):

NSCs differentiation on nanostructured rGO microfibers was also assessed by the gene expressions using qPCR at day 15. The total RNA was extracted from the cells using the RNeasy Plus Mini Kit (Qiagen), according to the manufacturer’s instructions. The RNA samples were reverse-transcribed into cDNA for qPCR using the PrimeScriptTM reagent kit with gDNA Eraser (Takara). qPCR was performed with SYBR Premix Ex TaqTM with ROX (Takara), according to the manufacturer’s instructions. The signals were detected with an ABI 7500 Fast Real Time PCR system (Applied Biosystems) to analyze the expressions of Tuj1 and GFAP. The gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal standard. Information on the primers is provided in Supplementary Information Table S1.