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5 protocols using transscript rt reagent kit

1

Quantitative RT-PCR for Viral RNA Detection

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Total RNA from virus‐infected cells or lung tissues was extracted using an RNA isolation kit (Thermo Scientific). First‐strand cDNA was synthesized from 1 µg of total RNA using a TransScript RT reagent kit (TransGen). Uni‐12 primer was used for the detection of influenza vRNA, and oligo dT and random primers were used for detecting host and viral genes. Generated cDNA was subjected to qPCR in a 20 µL reaction volume using FastStart Universal SYBR Green master mix (Roche). Human β‐actin and mouse GAPDH genes were amplified for normalization in qPCR. Reactions were conducted in triplicates, and the data were analysed using the 2−ΔΔCt method. qPCR primers used in this study are provided in the Table S1 (Supporting Information).
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2

Quantitative analysis of mRNA and miRNA

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Total RNA was isolated from tissue and cell specimens using Trizol (TaKaRa, China), and total RNA was extracted according to the manufacturer’s instructions. The RNA concentration was measured with a BioSpectrometer (Eppendorf, Germany). The RNA samples were reversely transcribed into cDNA using the TransScript RT reagent Kit (TransGen, China). QRT-PCR was performed with FastStart Universal SYBR Green Master (ROX) (Roche, USA). β-actin and U6 were used to normalize the level of mRNA and miRNA expression, respectively. β-actin primers were 5′-CTGGAACGGTGAAGGTGACA-3′ and 5′-AAGGGACTTCCTGTAACAATGCA-3′; PHLPP2 primers were 5′-CCAATGAGCAAGGACAGGAT-3′ and 5′-GGTCCTCTGGTTCCATCTGA-3′. The Bulge-Loop miRNA qRT-PCR Primer kit (RIBOBIO, China) was used for detecting miR-27a expression. QRT-PCR was performed using the CFX96 Real-Time system (Bio-Rad, USA), and the data were analyzed using the 2∆∆CT method.
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3

RNA Extraction and cDNA Synthesis Protocol

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Total RNA was extracted using the E.Z.N.A.® Plant RNA Kit (Omega Bio-tec, USA) according to the manufacturer's protocol. The quantity and quality of the RNA were analyzed by 1.2% (w/v) agarose gel electrophoresis and a ScanDrop® Spectrophotometer (Analytik Jena AG, Jena, Germany). cDNA was synthesized by the TransScript® RT Reagent Kit (TransGen Biotech, Peking, China) according to the manufacturer's protocol. The cDNA was diluted 10-fold with RNAase-free water for qRT-PCR.
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4

Comprehensive Transcriptional Analysis of Cellular Metabolism

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Total RNA was extracted from cells using RNAiso Plus (TaKaRa, Japan, 108‐95‐2) and isolated according to the manufacturer's instructions. The RNA concentration and purity were measured by a BioSpectrometer (Eppendorf, Germany); 2 μg total RNA was reversely transcribed into cDNA using the TransScript RT reagent Kit (TransGen, China, AE301). According to the manufacturer's instructions, qRT‐PCR was performed with FastStart Universal SYBR Green Master (Vazyme, China, Q111) using a Gene Amp 9600 PCR system (Perkin‐Elmer, Waltham, MA). The relative amount of cDNA was analyzed using the 2−ΔΔCT method. The primers for qRT‐PCR used in this study were as follows: PDHA1‐Forward: CTTACCGCTACCATGGACACAGCATG,
Reverse: CTCCTTTAATTCTTCAACACTTGCAAGA; HK2‐Forward: GAGCCACCACTCACCCTACT, Reverse: CCAGGCATTCGGCAATGTG; PFK2‐Forward: ATTGCGGTTTTCGATGCCAC, Reverse: GCCACAACTGTAGGGTCGT; IDH1‐ Forward: TTGGCTGCTTGCATTAAAGGTT, Reverse: GTTTGGCCTGAGCTAGTTTGA; CS‐Forward: GAGCAGGCCAGAGTTAAGAC, Reverse: AAAATAAGCCCTCAGGTAGG; LDHA‐Forward: AAACGCGCCTTAATTTAGTCCA, Reverse:CAGCCGCTTCCAATAATACGG; PGC1α‐Forward: GTAAATCTGCGGGATGATGG, Reverse: AGCAGGGTCAAAATCGTCTG; SIRT1‐Forward: TGCCATCATGAAGCCAGAGA, Reverse: AACATCGCAGTCTCCAAGGA; and GAPDH‐Forward:CAAGAAGGTGGTGAAGCAGG, Reverse: CCACCCTGTTGCTGTAGCC.
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5

Virus RNA Extraction and qPCR Analysis

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Total RNA from virus-infected cells was extracted using an RNA isolation kit (Thermo Scientific, Waltham, MA, USA). First-strand complementary DNA was synthesized from 1 μg of total RNA using a TransScript RT reagent kit (TransGen, Beijing, China), and oligo dT were used for detecting host genes. Generated cDNA was subjected to qPCR in a 25 μL reaction volume using FastStart Universal SYBR Green master mix (Roche, Shanghai, China). Human GAPDH genes were amplified for normalization of the cDNA amount used in qPCR. Reactions were conducted in triplicate, and the data were analyzed using the 2−ΔΔCt method.
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