Real-time PCR was performed as previously reported [25 (link)]. Total RNA was isolated from cells using a standard Trizol (Invitrogen) extraction protocol using an RNeasy mini extraction kit (Qiagen) according to the manufacturer’s instructions. RNA was converted to cDNA using a high-capacity cDNA reverse transcription kit (Ambion). Real-time PCR was performed on a 7500 Fast RT-PCR instrument (Applied Biosystems) using 20 ng/uL cDNA, TaqMan PCR master mix (Applied Biosystems), and gene-specific TaqMan probes (Applied Biosystems) against TAP1 (Mm00443188_m1), EHD1 (Mm01236839_m1), IRF1 (Mm01288580_m1), IRF7 (Mm00516793_g1), NFkB (Mm00476361_m1), IL1B (Mm00434228_m1), Arg1 (Mm00475988_m1), GAPDH (Mm99999915_g1), and HPRT (Mm03024075_m1). For each RNA sample, each primer set was run in triplicate. Gene expression was normalized to the internal control GAPDH (for BV2 microglia) or HPRT (for primary microglial cells), and relative expression was calculated for each gene using the 2ΔΔCT method after normalizing to the control sample.
7500 fast rt pcr instrument
Manufactured by Thermo Fisher Scientific
The 7500 Fast RT-PCR instrument is a real-time PCR system designed for fast and accurate quantification of nucleic acids. It features a 96-well block, a sensitive optical detection system, and advanced software for data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Taqman pcr master mix, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Magna merip m6a kit, by Merck Group (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini extraction kit, by Qiagen (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Dnase, by Merck Group (1 mentions)
Gene specific taqman probe, by Thermo Fisher Scientific (1 mentions)
Quantifast sybr green rt pcr kit, by Qiagen (1 mentions)
Mrna purification kit, by Thermo Fisher Scientific (1 mentions)
Anti m6a antibody, by Abcam (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 4, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
8 protocols using 7500 fast rt pcr instrument
1
Real-time PCR Gene Expression Analysis
2
Quantifying m6A Levels in LUAD Cells
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The MeRIP assay was used to determine the m6A levels of the target gene ADIRF in A549 and H157 cells.33 (link) Total RNA was isolated from A549 and H157 cells. Stable high-expressing ALKBH5 and LUAD cells transfected with a vacant vector were used as controls (A549/vector, H157/vector). The obtained mRNA was purified using DNase (Sigma) and fragmented into ~ 100 nucleotides. m6A primary antibody (10 μg, Abcam, ab151230) linked to Magna ChIP protein/G magnetic beads was co-immunoprecipitated with the fragmented mRNA for 2 h at 4°C according to the protocols of the Magna MeRIP™ m6A kit (17–10499, Millipore, Germany). m6A RNA elution and purification were performed using an eluent containing N6-methyladenosine 5’-monophosphate sodium salt (twice, 1 h), followed by overnight precipitation in ethanol (−80°C). qRT-PCR was then performed on a 7500 Fast RT-PCR instrument (Applied Biosystems) to analyze the mRNA levels of m6A sites.
3
Quantitative RT-PCR Gene Expression Analysis
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CNS-MP RNA extracted in Trizol was purified for qRT-PCR [18 (link), 24 (link)]. RNA was reverse-transcribed to cDNA (Ambion), and qRT-PCR was performed (7500 Fast RT-PCR instrument, Applied Biosystems) using cDNA, TaqMan PCR Master Mix and gene-specific TaqMan probes (Applied Biosystems) against Kcna3 (Mm00434599_s1), Kcnj2 (m00434616_m1), Ptgs2 (Mm00478374_m1), and Hprt (Mm03024075_m1) in duplicate. Relative gene expression was normalized to Hprt and calculated using the 2ΔΔCT method [25 (link)].
4
Quantitative RT-PCR for Metallothionein Expression
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RT-PCR was performed by using the QuantiFast SybrGreen RT PCR Kit (Qiagen) according to the protocol. The reactions were conducted with a 7500 Fast RT-PCR instrument (Applied Biosystems, Foster City, CA). The housekeeping gene 18S was used as a reference gene. Primer’s sequences for metallothionein I and II and 18S known from previous studies by Świergosz-Kowalewska et al. (2007 (link)) were synthesised by Genomed (Warsaw, Poland) and then used for gene expression analyses. To amplify the target genes, 20 µg of RNA from each tissue sample was used. At the end of each RT-PCR reaction, the melting curve procedure was performed to check for possible unspecific products. All the reactions were run in three replicates. On each plate one identical sample (SS—RNA isolated from one of random chosen bank vole liver samples) was run to standardise readings between plates. The results of MT I, MT II, and 18S gene expressions were determined as CT (cycle number) values.
5
m6A Enrichment Analysis of ANGPTL3
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The Magna MeRIP™ m6A kit (Millipore, USA) was utilized to determine the enrichment level of m6A on ANGPTL3 sequences. Briefly, total RNA was obtained from AGS and HGC27 cells transfected with si-METTL3, using cells transfected with si-NC as control. Subsequently, mRNA Purification Kit (Invitrogen) and fragmentation buffer were used separately to purify and fragment total RNA (50 µg) in each sample. Anti-m6A antibody (Abcam) or anti-IgG was co-immunoprecipitated for 2 h at 4 °C with the obtained purified mRNA in IPP buffer, which contains pretreated protein A/G magnetic beads (Thermo Scientific). Finally, the corresponding m6A enrichment levels in the co-precipitated RNA samples were analyzed and calculated on a 7500 Fast RT-PCR instrument (Applied Biosystems).
6
Quantitative PCR Analysis of mtDNA and Gapdh
7
SARS-CoV-2 Detection Using RT-PCR
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RNA was extracted with MagMAX viral/pathogen (ThermoFisher) nucleic acid isolation kit using KingFisher Flex instrument for automated sample purification. Extracted RNA was reverse transcribed to cDNA and amplified/detected using the EUA TaqPath COVID-19 Combo Kit (ThermoFisher) on an Applied Biosystems 7500 Fast RT-PCR instrument. The assay contains probes to detect and amplify three SARS-CoV-2 specific target genes: ORF1ab, N gene, and S gene. If three targets are detected or if any 2 of the 3 target genes are detected, the test reports a positive result.
8
Quantifying LUAD-associated Gene Expression
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Total RNA was isolated from human tissue samples (48 pairs of LUAD and paracancerous tissues) and target LUAD cells (A549, H157, H460, H1299 and PC9) using an RNA extraction kit (TRIzol, Invitrogen, USA). Overall, 500 ng of target RNA samples (ADIRF, ALKBH5, and YTHDC2) was directly reverse transcribed to cDNA using reverse transcriptase (Superscript IV, Invitrogen). qRT-PCR was performed using a PCR kit (SYBR® Premix Ex Taq™, DRR041S, Takara) on a 7500 Fast RT-PCR instrument (Applied Biosystems) to process the qRT-PCR step. The qPCR reaction was performed in a 25 µL volume consisting of 12.5 µL SYBR® Premix Ex Taq™, 1 µL forward/reverse primer (10 µmol/L), 2 µL cDNA, and 8.5 µL ddH2O. Thermal cycling parameters were set as follows: initial denaturation (95°C for 5 min), followed by 40 cycles of denaturation (95°C for 5s; 60°C for 30s). The ADIRF, ALKBH5, and YTHDC2 levels were normalized to those of β-actin. The relative expression levels of target genes were calculated using the 2–∆∆Ct method. The primer sequences for the specific genes were as follows:
ADIRF-F: 5’-AGACTGCTAACCAGGCCTCT-3’
ADIRF-R: 5’-CCGAATTTGGGAGGCTAGGA-3’
ALKBH5-F: 5’-CGGCGAAGGCTACACTTACG-3’
ALKBH5-R: 5’-CCACCAGCTTTTGGATCACCA-3’
YTHDC2-F: 5’-AGGACATTCGCATTGATGAGG-3’
YTHDC2-R: 5’-CTCTGGTCCCCGTATCGGA-3’
β-Actin-F: 5’-CTCCATCCTGGCCTCGCTGT-3’
β-Actin-R: 5’-GCTGTCACCTTCACCGTTCC-3’
ADIRF-F: 5’-AGACTGCTAACCAGGCCTCT-3’
ADIRF-R: 5’-CCGAATTTGGGAGGCTAGGA-3’
ALKBH5-F: 5’-CGGCGAAGGCTACACTTACG-3’
ALKBH5-R: 5’-CCACCAGCTTTTGGATCACCA-3’
YTHDC2-F: 5’-AGGACATTCGCATTGATGAGG-3’
YTHDC2-R: 5’-CTCTGGTCCCCGTATCGGA-3’
β-Actin-F: 5’-CTCCATCCTGGCCTCGCTGT-3’
β-Actin-R: 5’-GCTGTCACCTTCACCGTTCC-3’
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