Total RNA was isolated from cell lines using the mirVana kit (Ambion, Austin, TX) according to the manufacturer's instructions and 10 ng of total RNA was reverse transcribed using the TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative PCR (qPCR) was performed on the Agilent 3000P system using the human miR-34a and U6 miRNA TaqMan expression assays (Applied Biosystems). Relative miR-34a expression was determined using the gene comparative CT method. For gene expression analysis, 200 ng of total RNA was reverse transcribed using ThermoScriptTM RT-PCR system for first strand cDNA synthesis (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Gene expression was then determined by qPCR using the KiCq Start SYBR Green kit (Sigma, St. Louis, MO). The primers sequences used for gene expression SYBR Green qPCR are listed in Supplemental Table 1 .
Thermoscript rt pcr system for first strand cdna synthesis
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ThermoScript RT-PCR System is a tool for first-strand cDNA synthesis. It is designed to generate high-quality cDNA from RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Strata gene mx3000p real time pcr system, by Promega (2 mentions)
Gotaq qpcr master mix, by Promega (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
7900ht real time pcr system, by Thermo Fisher Scientific (2 mentions)
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirvana kit, by Thermo Fisher Scientific (1 mentions)
Kicqstart sybr green kit, by Merck Group (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Megabace system, by GE Healthcare (1 mentions)
Trizol protocol, by Thermo Fisher Scientific (1 mentions)
Rna purification kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Sensimix sybr kit, by Meridian Bioscience (1 mentions)
6100 nucleic acid prepstation, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Transcriptor high fidelity cdna synthesis kit, by Roche (1 mentions)
15 protocols using thermoscript rt pcr system for first strand cdna synthesis
1
Quantitative Analysis of miR-34a and Gene Expression
2
Quantification of Gene Expression in Adipose Tissue
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Total RNA was isolated using TRIzol® Reagent (Invitrogen, Cat# 15596-026) according to the manufacturer's recommendations. RNA concentrations were quantified and reverse-transcribed using ThermoScriptTM RT-PCR System for First-Strand cDNA Synthesis (Invitrogen, Cat# 11146-016). Gene expressions were detected using GoTaq qPCR Master Mix (Promega, Cat# A6001) in Strata gene MX3000P Real-Time PCR system (Genetimes, China). Relative gene expression levels were calculated by ΔΔCt and compared with GAPDH as internal control.
Primers were designed using Primer 5.0 software: PPARγ (NM_001145366; forward: 5′-GTCACACTCTGACAGGAGCC-3′; reverse: 5′-CAC CGCTTCTTTCAAATCTTGT-3′), GAPDH (NM_017008; forward: 5′-AAGGGCTCATGACCACAGTC-3′; reverse: 5′-CAGGGATGATGTTCTGGGCA-3′), OBRb (AF287268; forward: 5′-TCCAGGTGAGGAGCAAGAG-3′; reverse: 5′-TTCAGCGTAGCGGTGATG-3′), and STAT3 (NM_012747; forward: 5′-TATCTTGGCCCTTTGGAATG-3′; reverse: 5′-GTTGTAGGACCATAGGGGTG-3′). Each reaction mixture (25 μL total volume) contained 12.5 μL Maxima SYBR Green Master mix (2X), 0.3 μM of each primer, nuclease-free water, and 20 ng template DNA. Amplification of each gene was performed in duplicate runs and PCR conditions were 95°C for 10 min, followed by 40 cycles of 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C.
Primers were designed using Primer 5.0 software: PPARγ (NM_001145366; forward: 5′-GTCACACTCTGACAGGAGCC-3′; reverse: 5′-CAC CGCTTCTTTCAAATCTTGT-3′), GAPDH (NM_017008; forward: 5′-AAGGGCTCATGACCACAGTC-3′; reverse: 5′-CAGGGATGATGTTCTGGGCA-3′), OBRb (AF287268; forward: 5′-TCCAGGTGAGGAGCAAGAG-3′; reverse: 5′-TTCAGCGTAGCGGTGATG-3′), and STAT3 (NM_012747; forward: 5′-TATCTTGGCCCTTTGGAATG-3′; reverse: 5′-GTTGTAGGACCATAGGGGTG-3′). Each reaction mixture (25 μL total volume) contained 12.5 μL Maxima SYBR Green Master mix (2X), 0.3 μM of each primer, nuclease-free water, and 20 ng template DNA. Amplification of each gene was performed in duplicate runs and PCR conditions were 95°C for 10 min, followed by 40 cycles of 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C.
3
Cloning and Sequencing of Ago2-bound RNAs
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The pre-Agosh RNA samples obtained from the Ago2 immunoprecipitation were cloned and sequenced. For this purpose, the small RNAs were 3’ polyadenylated using the A-Plus Poly(A) Polymerase Tailing Kit (Epicentre Biotechnologies) according to manufacturer’s protocol. The reaction was stopped by acid-phenol:chloroform extraction followed by ethanol precipitation. Subsequently, the polyadenylated RNAs were reverse transcribed into cDNA, using the ThermoScript RT-PCR System for first-strand cDNA synthesis (ThermoFisher Scientific), followed by PCR amplification. PCR products were separated on size via agarose gel electrophoresis, purified and subsequently used for TOPO-TA cloning (TOPO-TA cloning kit, Life Technologies). TA clones were sequenced with the T7 and m13RP primer using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems).
4
3'RACE PCR for HTR4b transcript identification
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1 µg total RNA from human ileum and colon were reverse transcribed using the ThermoScript RT-PCR System for First-Strand cDNA Synthesis (Thermo Fisher Scientific) according to the manufacturer’s instructions. 3′RACE PCRs specific for HTR4b were performed using the HotStarTaq DNA Polymerase protocol. First PCR was run for 15 cycles with 2 min elongation time using 1 µl of the Thermoscript cDNA product as template. 1 µl product of the first PCR run was taken as template for the following nested PCR, which was run for another 30 cycles with 2 min elongation time per cycle, respectively. Primer sequences given in Supplementary Table S1 . PCR products were analysed on a 1.5% agarose gel, cloned into the pSTBlue-1 AccepTor Vector (Merck Millipore, Billerica, Massachusetts) according to the manufacturer’s instructions and sequence verified with the MegaBACE system (GE Healthcare, Little Chalfont, United Kingdom).
5
Transcriptome Analysis of Insect Antennae
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Antennae were removed from the males and females under a stereo microscope (Olympus SZ Series Zoom, Olympus, Tokyo, Japan) and stored at -80°C until RNA isolation. Total RNA from pools of antennae (50 pairs from each sex of R. nubila, 100 pairs from male E. semipurpurella, and 100 pairs from each sex of L. capitella) was extracted using the Trizol protocol (Thermo Fisher Scientific, Carlsbad, CA, United States), and subsequently purified using an RNA Purification Kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. The concentration and quality of the RNA were analyzed using a Nanodrop2000 (Thermo Scientific, Saveen Werner, Malmö, Sweden). First-strand cDNA was synthesized from 1 μg of DNAse-treated total RNA using the ThermoScript RT-PCR system for First-Strand cDNA Synthesis (Thermo Fisher Scientific) following the manufacturer’s instructions, except that both oligo-dT primers and random hexamers (1 μL of each) were used in the 20 μL reaction mixtures. The first-strand cDNA library was then used for cloning of chemosensory genes (see section “PCR Confirmation and RACE-PCR Amplification”).
6
Transcriptome Analysis of AM and HIF-1α
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Total RNA was extracted using the 6100 Nucleic Acid Prepstation (Applied Biosystems, Darmstadt, Germany) following the manufacturer's protocol. In all, 150 ng total RNA was reverse transcribed with the ThermoScript RT-PCR System for First-Strand cDNA Synthesis (Invitrogen, Darmstadt, Germany), SuperScript III Reverse Transcriptase (Invitrogen) or Transcriptor High Fidelity cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. Quantitative RT-PCR was performed in ABI Prism 7000 Sequence Detection System (Applied Biosystems) using the SensiMix SYBR Kit (Bioline, Luckenwalde, Germany) or Taqman Gene Expression Assay (Invitrogen). The mRNA levels were normalized to human L28 mRNA. Following primers and assays were used for quantitative RT-PCR: human AM forward, 5′-GGA TGC CGC CCG CAT CCG AG-3′ human AM reverse, 3′-GAC ACC AGA GTC CGA CCC GG-5′ human L28 forward, 5′-ATG GTC GTG CGG AAC TGC T-3′ human L28 reverse, 5′-TTG TAG CGG AAG GAA TTG CG-3′ Taqman Gene Expression Assay for HIF-1α (Hs00936368_m1).
7
Transcriptional Analysis of Babesia bigemina
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RNA from B. bigemina infected erythrocytes, induced sexual stages of the parasite, pool of infected and uninfected ticks 0 h and 72 h post-repletion and from the midgut of infected ticks, 72 h post-repletion was purified using TRIzol (Invitrogen). Then, 1 μg of total RNA was reverse-transcribed with ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis (Invitrogen), and used for amplification with Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) following the supplier’s instructions. Two μl of cDNA of each sample was used for PCR. The oligonucleotides used for amplification of 375 bp hap2 were pF (5′-GAC AAA TTC ACC GAC ACG TC-3′) and pR (5′-CTA CAC CTC GTC GCT ATG-3′). Amplification conditions for hap2 were: an initial denaturing step at 94 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 58 °C for 30 s and 72 °C for 40 s, with a final extension step at 72 °C for 7 min and 4 °C indefinitely. To obtain a 155 bp amplicon of the gapdh as a reference gene, we designed a pair of primers from the sequence BBBOND_0202520: forward (5′-CCG CAC CAT CAA ATT GTA C-3′) and reverse (5′-CGG AGA TGA TGA CCA ACT TG-3′). RNA from uninfected erythrocytes and uninfected ticks was used as negative control.
8
RNA Isolation, cDNA Synthesis, and qPCR Analysis
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Total RNA was isolated from tissue or cells using TRIZOL reagent (Invitrogen). The cDNA synthesis was performed using ThermoScript RT-PCR System for First-Strand cDNA Synthesis (Invitrogen). Real-time PCR was performed with the 7900HT Real-Time PCR system (Applied Biosystems) using SYBR Green reagents. All analysis was normalized against the cycle threshold number of GAPDH or cyclophilin genes, then calculated using the following equation: RCN = 2−ΔCt ×100, where ΔCt is the Ct(target) – Ct(reference). The sequences of all the probes are available upon request.
9
RNA Isolation and qRT-PCR Analysis
10
Quantitative Real-Time PCR Analysis
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Total RNA was isolated using TRIzol® Reagent (Invitrogen, Cat#15596-026) according to the manufacturer’s recommendations. RNA concentrations were quantified and reverse-transcribed using ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis (Invitrogen, Cat#11146-016). Gene expressions were detected using GoTaq qPCR Master Mix (Promega, Cat#A6001) in Strata gene MX3000P Real-Time PCR system (Genetimes, China). Relative gene expression levels were calculated by △△Ct and compared with GAPDH as internal control.
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