RNA was extracted with TRIzol reagent (Sigma-Aldrich) in TFH cells (CXCR5+PD1+CD4+T cells) sorted from the spleen, BALF, or lung. Complementary DNA (cDNA) was synthesized using the PrimeScript™ RT Master Mix (Perfect Real Time; TaKaRa, Osaka City, Osaka Prefecture, Japan). An ABI Q6 Flex Real-time PCR system (ThermoFisher Scientific) was used for quantitative PCR with primers from Applied Biosystems (Carlsbad, CA, USA). The Glut1 gene-specific primers used in this study are as follows: forward primer, 5′-cagctgtcgggtatcaatgc-3′; reverse primer, 5′-tccagctcgctctacaacaa-3′. The Sdha gene-specific primers used in this study are as follows: forward primer, 5′-tgctgggtacttgaatccct-3′; reverse primer, 5′-atgaacgtagtcggtaaccac-3′. The individual gene expression was calculated and normalized to the expression of Hprt. The primers used for Hprt were as follows: forward primer, 5′-agtacagccccaaaatggttaag-3′; reverse primer, 5′-cttaggctttgtatttggcttttc-3′. To determine the relative quantities, SYBR® Premix ExTaqTM (Perfect Real Time, TaKaRa) was used. The results were analyzed with an ABI Q6 Flex Real-time PCR system (ThermoFisher Scientific), as described previously [26 (link)].
Abi q6 flex real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland
The ABI Q6 Flex Real-time PCR system is a laboratory instrument designed for real-time polymerase chain reaction (PCR) analysis. It provides precise temperature control and fluorescence detection capabilities to enable quantitative gene expression analysis and detection of nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
2pcr master mix, by Roche (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taqtm, by Takara Bio (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Arevertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Microspectrophotometer, by Tiangen Biotech (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
5 protocols using abi q6 flex real time pcr system
1
Quantitative PCR analysis of T cell markers
2
qRT-PCR Profiling of mRNA and tsRNA
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For mRNA expression, RNA was reverse-transcribed into cDNA by the RevertAid™ First Strand cDNA Synthesis Kit, with DNase I (K16225; Thermo Fisher, Waltham, MA, US), using random primers. For tsRNAs expression, RNA was pretreated with aRevertAid™ First Strand cDNA Synthesis Kit, with DNase I (K16225; Thermo Fisher, Waltham, MA, USA) using stem-loop reverse transcription primers. Next, the amplification reaction was carried out with cDNA as the template using the 2PCR Master Mix (Roche, Basel, Switzerland) on the ABI Q6 Flex Real-time PCR system (Applied Biosystems, Waltham, MA, USA). All primer’s sequences were synthesized by GenePharm (China) and shown in Table S1 . The 2−ΔΔCT method was used for normalizing gene expression relative to GAPDH or U6.
3
Quantifying Gene Expression in Lens Cells
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Total RNA was extracted from crystalline lens samples and HLE-B3 cells using TRIzol (Invitrogen). The concentration and purity of total RNA were measured using a microspectrophotometer (Tiangen, Beijing, China). High-quality RNA was stored at −80 °C for subsequent RT-qPCR and RNA sequencing. Next, reverse transcription was conducted using a RevertAid™ First Strand cDNA Synthesis Kit (K16225; Thermo Fisher, Waltham, MA, USA). cDNA amplification was performed by 2 × PCR Master Mix (Roche, Basel, Switzerland) on the ABI Q6 Flex Real-time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression was calculated using the 2−ΔΔCT method. All primers used in this study are shown in Table S2 .
4
RNA Isolation and RT-qPCR for Adipose Tissue
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RNA isolation and RT-qPCR were performed as described previously [24 (link)]. Briefly, according to manufacturer's instructions, total RNA of BAT was extracted using TissueLyser II with TRIzol (#15596, Invitrogen Corporation, Carlsbad, CA, USA), subsequently precipitated with isopropanol and subjected to clean-up using 75% ethanol. RNA concentrations were measured using the NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Reverse transcription was conducted using a commercial kit (#RR037A, Takara Biomedical Technology, Co., Ltd., Dalian, China). SYBR Premix Ex Taq (#RR420A, Takara Biomedical Technology, Co., Ltd., Dalian, China) was used for qPCR. Amplification of cDNA and fluorescence measurement was performed using an ABI Q6 Flex Real-time PCR System (Applied Biosystems Inc., Foster City, CA). Each tissue sample was measured in triplicate and resulting average was used for further statistical analysis. Primers were designed using Primer Express 4 and Primer Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) software and synthesized by Life Technologies (Shanghai, China). The sequences are shown in Table S2 . The expression level of 36B4 was used as the loading control and the relative expression of genes is shown as fold-change over control (2-ΔΔCt).
5
Sanguinarine Inhibits PRRSV Replication
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To probe the effect of sanguinarine on the proliferation of PRRSV, the abundance of PRRSV ORF7 and nsp9 RNA transcripts was determined via RT-qPCR. Briefly, equal amounts of RNA were reverse transcribed into cDNA with oligo (dT) primers and Transcriptor First Strand cDNA Synthesis Kits (Roche, Basel, Switzerland). Then, the cDNA was used for qPCR in triplicates with primers (Table 1 ) specific to PRRSV ORF7 and nsp9 genes, respectively. The qPCR was conducted with Power SYBR green PCR master mix (Applied Biosystems, Waltham, MA, USA) in an ABI Q6 Flex real-time PCR system (Applied Biosystems).
To explore the role of sanguinarine in the replication stage of PRRSV infection, the PRRSV negative-sense RNA level was quantified as well. Especially, RNA was reverse transcribed into cDNA with 5′UF primers (Table 1 ), which bind to the viral negative-sense RNA, and the primers (Table 1 ) specific to PRRSV 5′UTR were used in qPCR.
To explore the role of sanguinarine in the replication stage of PRRSV infection, the PRRSV negative-sense RNA level was quantified as well. Especially, RNA was reverse transcribed into cDNA with 5′UF primers (
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