Eastep super total rna extraction kit

To quantify miR-9 in cells or tissues, total RNA was purified using an Eastep Super total RNA extraction kit (catalogue no. LS1040m, Promega) following the manufacturer’s protocol for retaining small RNA, reverse transcribed using a miRNA 1st Strand cDNA Synthesis Kit (catalogue no. MR101-01, Vazyme Biotech), and PCR amplified using a ChamQ Universal SYBR qPCR kit (catalogue no. Q711-02/03; Vazyme Biotech). The stem-loop primers and qPCR primers for miR-9 quantification were purchased from RiboBio. To quantify transcripts, RNA was isolated using an Eastep Super total RNA extraction kit (catalogue no.LS1040m, Promega), reverse transcribed using a HiScript II Q Select RT supermix kit (catalogue no. R233-01; Vazyme Biotech), and PCR amplified using a ChamQ Universal SYBR qPCR kit (catalogue no. Q711-02/03; Vazyme Biotech). The analyzed transcript levels were normalized to GAPDH transcript levels. To quantify viral genomes, DNA was isolated using a DNA isolation kit (catalogue no.DC102-01; Vazyme Biotech) and qPCR was conducted using the ChamQ Universal SYBR qPCR kit (catalogue no. Q711-02/03; Vazyme Biotech). Viral genome levels were normalized to mouse Adipsin gene levels. Serially diluted total DNA, total RNA, or synthetic miR-9 was used to generate standard curves. PCR primer sequences are listed in Supplementary Table 4.

Total RNA was extracted from maize leaves using an EastepTM Super Total RNA Extraction Kit (Promega, Shanghai, China). First-strand cDNA was synthesized using 1 μg of total RNA, oligo (dT18) primer and random primers, and M-MLV reverse transcriptase (Vazyme, Nanjing, China) in each 20 μL reaction. RT–qPCR was performed using the ChamQ Universal SYBR RT–qPCR Master Mix (Vazyme, Nanjing, China) on an ABI 7500 FAST Real-Time System (Applied Biosystems, Foster City, CA, USA). The specific primers (Table S10) were designed using Primer 5 software [86 (link)]. The expression of the ZmUBI (XM_008647047) gene served as an internal control, and relative gene expression levels were calculated using the 2^−ΔΔCt method.

Total RNAs were extracted with Eastep^TM Super Total RNA Extraction Kit (Promega, LS1040, Beijing, China) following the manufacturer’s instructions, and then reverse-transcribed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, AT311, Beijing, China). The cDNA was diluted 10 times with sterilized distilled water. Quantitative RT-PCR (qRT-PCR) was conducted, according to the instructions of TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara, RR820A, Dalian, China), on a ABI QuantStuio 7 Flex RT-PCR instrument (Applied Biosystems, Foster City, CA, USA). Gene-specific primers for qRT-PCR were shown in Table S3. The relative expression levels of the target CCT genes were presented as fold-change, calculated using the comparative method.