Simply p total rna extraction kit

Six normal brain tissues, LGG, GBM tissues, which obtained from the Second Affiliated Hospital of Nanchang University, were utilized in this research. We isolated total RNA from brain tissues with the Simply P Total RNA Extraction Kit (Bioflux, Beijing, China) and then reverse-transcribed it into complementary DNA with HiScript III-RT SuperMix (Vazyme, Nanjing, China). Then, relative expression of lncRNAs was normalized to GAPDH, and fold change was inspected by employing the 2^−∆∆CT method. The primer sequences were displayed in Supplementary Table S5.

Total cellular RNA was extracted using the Simply P Total RNA Extraction Kit (BioFlux, China) according to the manufacturer's instructions. Total RNA (1 μg) was reverse-transcribed to cDNA using the PrimeScript™ II First-Strand cDNA Synthesis Kit (Takara, China). The synthesized cDNA was subjected to qPCR performed in triplicate using a LightCycler^® 480 II real-time PCR instrument (Roche, Switzerland) and SYBR Green PCR mix (Life Technologies, USA) according to the manufacturer's instructions. All the data were acquired and analyzed using LightCycler^® 480 II software 1.5 based on the cycle threshold (^ΔΔCT) method (25 (link)). GAPDH served as the internal control. The amplification efficiency of qPCR primers ranged from 85.83 to 106.38%. The primers used in this assay were designed using Primer Premier 5 software and are listed in Table 1.

qRT-PCR was performed to determine the expression levels of GATA-3, T-bet, and ANXA1. Total RNA extraction was performed using simply P total RNA extraction kit (Bioflux, Europe). The cDNA synthesis was performed using All-in-One™ First-Strand cDNA Synthesis Kit (GeneCopoeia, Maryland, USA). cDNA was used to set up a quantitative real-time PCR (qPCR) reaction using the All-in-One qPCR Mix (GeneCopoeia). The expression levels of GATA-3, ANXA1, and T-bet were normalized to actin. The primer sequences were as follows: mouse GATA-3 forward primer: 5′-GCTGGATGGCGGCAAAG-3′, mouse GATA-3 reverse primer: 5′-GTGGGCGGGAAGGTGAA-3′, mouse ANXA1 forward primer: 5′-AAGGTGGTCCTGGGTCAGC-3′, mouse ANXA1 reverse primer: 5′-TGAGCATTGGTCCTCTTGGT-3′, mouse Tbx21/T-bet forward primer: 5′-ATGTTCCCATTCCTGTCCTTCA-3′, mouse Tbx21/T-bet reverse primer: 5′-AAATGAAACTTCCTGGCGCATC-3′, mouse actin forward primer: 5′-CATCCTGCGTCTGGACCTGG-3′, and mouse actin reverse primer: 5′-TAATGTCACGCACGATTTCC-3′. All protocols were carried out according to the manufacturer's instructions. Each sample was run in triplicate.

Total RNA was extracted from Vero CCL81 cells by using a Simply P Total RNA Extraction Kit (Bioflux, China) and reverse transcribed into cDNA using HiScript^® II Q RT SuperMix (Vazyme, China), following the manufacturer’s instructions. The quantitative real-time PCR assay for quantifying the PEDV genome was carried out by using the following primer and probe sequences: PEDV forward primer: 5’- CGTACAGGTAAGTCAATTAC-3’; PEDV reverse primer: 5’-GATGAAGCATTGACTGAA-3’; and PEDV Taq-Man^® probe: FAM-TTCGTCACAGTCGCCAAGG-TAMRA. Quantitative real-time PCR was performed using Hieff UNICON^® qPCR TaqMan Probe Master Mix (YENSEN, China). Each reaction was performed in triplicate.

Total RNA was extracted from cells with the Simply P Total RNA Extraction Kit (Bioflux, Beijing, China) and reverse-transcribed to synthesize cDNA with HiScript III-RT SuperMix for qPCR (Vazyme, Nanjing, China). The ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for qPCR. The primer sequences were as follows: the forward HSPA6 primer was 5′-CGT​GCC​CGC​CTA​TTT​CAA​TG-3′, the reverse HSPA6 primer was 5′-AAA​AAT​GAG​CAC​GTT​GCG​CT-3′; the forward GAPDH primer was 5′- GAA​CGG​GAA​GCT​CAC​TGG-3′, and the reverse GAPDH primer was 5′- GCC​TGC​TTC​ACC​ACC​TTC​T-3′. RNA samples were sequenced and analyzed by Shanghai Majorbio Biotechnology Co., Ltd. and Shanghai Jiayin Biotechnology Co.; Ltd. RNA-seq transcriptome library was prepared following TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA, United States) using 1 μg of total RNA.

Cells were infected with TGEV, PEDV, and PDCoV at an MOI of 0.02, 0.2 and 1 and collected at 24 hpi. RNA was extracted using Simply P total RNA extraction kit (BioFlux, Beijing, China) according to the manufacturer’s instructions in 24-well plates. Total RNA (2 μg) was reverse transcribed into complementary DNA (cDNA) using M-MLV reverse transcriptase (Takara, Otsu, Shiga, Japan) and an Oligo(dT)₁₅ primer (Takara, Otsu, Shiga, Japan). The cDNA was subsequently subjected to real-time quantitative PCR (qPCR) in triplicate by using FastStart Essential DNA Green Master (Roche, Basel, Switzerland). Primers based on the TGEV N gene, PEDV N and PDCoV N gene were synthesized for quantification of the virus genome in qPCR. The primers are shown in Table 1 and were created using the program Oligo 6. The results were analyzed based on the cycle threshold (ΔΔCT) method using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference for the relevant viral replication levels.