Total RNA was extracted using TRIzol Reagent (Life Technologies) according to manufacturer’s instructions. Reverse transcription was performed with 2-μg total RNA. Real-time PCR was performed using the ABI PRISM 7500 Sequence Detector (Applied Biosystems) and SYBR Premix Ex Taq II (TaKaRa, Kusatsu, Shiga Prefecture, Japan), according to manufacturer’s guidelines. All mRNA expression levels are presented as the mean ± standard deviation (SD) from two or three independent experiments. Melting curves of PCR products were assessed for quality control.
Abi prism 7500 sequence detector
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Japan
The ABI Prism 7500 Sequence Detector is a real-time PCR instrument designed for quantitative gene expression analysis. It utilizes fluorescence detection to monitor the amplification of DNA sequences during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (24 mentions)
Trizol, by Thermo Fisher Scientific (8 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (7 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (5 mentions)
Sybr green kit, by Qiagen (5 mentions)
Quantitect sybr green rt pcr kit, by Qiagen (4 mentions)
Starscript first strand cdna synthesis kit, by Transgene (4 mentions)
Primescript rt reagent kit, by Takara Bio (4 mentions)
Sybr green master mix, by Vazyme (4 mentions)
Sequence detector software, by Thermo Fisher Scientific (4 mentions)
Sybr select master mix, by Thermo Fisher Scientific (3 mentions)
Rneasy mini column, by Qiagen (3 mentions)
Qpcr rt kit, by Toyobo (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Rna pcr kit, by Takara Bio (2 mentions)
Random hexamer, by Takara Bio (2 mentions)
Sv total rna isolation system, by Promega (2 mentions)
76 protocols using abi prism 7500 sequence detector
1
Transcriptome Analysis by RT-qPCR
2
Quantification of miR-497 Expression
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Total RNA was extracted using TRIzol (Invitrogen, U.S.A.) according to the manufacturer’s instructions. Two micrograms of total RNA from each sample was reverse transcribed into cDNA using the RNA PCR Kit (Takara Biotechnology, Japan). The real-time quantitative PCR was performed on the ABI PRISM 7500 Sequence Detector (Applied Biosystems). Then, qRT-PCR was performed to quantify the expression level of miR-497 with SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. miR-497 expression normalized to U6 was calculated using the comparative Ct method formula: 2−ΔΔCt. Primers for amplification of U6 and miR-497 are listed in Table 1 .
3
Quantification of Regulatory T-cell Markers
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Cells were homogenized in TRIzol reagent (Sigma-Aldrich), and RNA extraction was performed using TaKaRa RNAiso reagent (TaKaRa). For reverse transcription, cDNA was synthesized using a PrimeScipt™ RT Master Mix (TaKaRa). The real-time PCR efficiency for each primer pair was calculated using standard curves generated through serial dilution of cDNA from Tregs. Primer sequences are shown in Table 1 . PCR reactions were performed using an ABI PRISM 7500 Sequence Detector (Applied Biosystems). The PCR conditions were set as follows: an initial incubation step for 30 s at 95°C followed by 40 cycles of 5 s at 95°C and 34 s at 60°C. The normalized expression values for each transcript were calculated as the quantities of FOXP3, TGF-β and IL-10 mRNA relative to the quantity of β-ACTIN mRNA using the 2−ΔΔCt method. All reactions were performed independently at least three times.
Primer sequences for real-time PCR.
| Gene | Forward (5′–3′) | Reverse (5′–3′) |
|---|---|---|
| β-ACTIN | ATCTGCTGGAAGGTGGACAGCGA | CCCAGCACAATGAAGATCAAGATCAT |
| FOXP3 | GTGGCCCGGATGTGAGAAG | GGAGCCCTTGTCGGATGATG |
| TGF-β | AACGAACTGGCTGTCTGC | CCTCTGCTCATTCCGCTTAG |
| IL-10 | GCTGGAGGACTTTAAGGGTTAC | ATGTCTGGGTCTTGGTTC |
FOXP-3, Forkhead box P3; IL-10, interleukin-10; TGF-β, transforming growth factor-β.
4
qPCR Validation of Differentially Expressed Proteins in FRDA
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The qPCR validation was done in 10 FRDA patients and 10 matched healthy controls in triplicate reactions. These samples were taken from different patients and control from those the proteomics results were obtained. Total RNA was reverse-transcribed into cDNA using a PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio Inc.). The mRNA levels of four differentially expressed proteins, actin α cardiac muscle 1 (ACTC1), pyruvate dehydrogenase E1 subunit β (PDHE1), caspase 8 (CAS8), and serotransferrin (TF) were assayed. Primers designing for ACTC1, PDHA1, CAS8, and TF were performed using Gene Bank Graphics database3 and Primer3 software4 . GAPDH was used as reference in order to normalize expression levels and to quantitate changes in gene expressions between the control and FRDA samples. The qPCR was run on the ABI Prism 7500 Sequence Detector (Applied BioSystems, Foster City, CA): 2 min at 50°C, 10 min at 95°C, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Gene and primer set with details are given in Table 2 . Changes in gene expression of the selected genes between FRDA and controls were determined by the comparative ΔΔCT method (Livak KJ, Schmittgen TD., 2001) using GAPDH as internal references.
5
Quantitative Analysis of RNA Expression
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Total RNAs were extracted from cells using the Trizol (Invitrogen, USA) reagent according to the manufacturer's instructions. The first-strand cDNA was synthesized using M-MLV (Promega, USA) following standard protocols. Dnd1, Bim, and GAPDH mRNA expression levels were detected using the specific primers and SYBR Green Master Mix (Biomics Biotechnology Inc., China). The miRNA first-strand cDNA was generated with miRNA reverse transcription kit (Abm, Canada). miRNA qRT-PCR kit and miR-221 primer were purchased from Abm. U6 snRNA was used as an endogenous quantity control for miRNA quantification. And qRT-PCR was performed on an ABI Prism 7500 Sequence Detector (Applied Biosystems, Life Technologies). Melting curve analysis was used routinely to check the specificity of amplification.
6
Osteosarcoma Genetic Expression Analysis
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Expression of caspase 3, NOTCH1, HEY1, HES1, and HES5 in osteosarcoma cells was determined at indicated times by RNA preparation and quantitative reverse transcription polymerase chain reaction (RT-PCR). Briefly, total cellular RNA was isolated from cells on six-well plates using TRIzol reagent following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). RNA quality was assessed by agarose gel electrophoresis, and complementary DNA (cDNA) was synthesized with random hexamer (TaKaRa, Osaka, Japan). The real-time RT-PCR analysis was carried out using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Valencia, CA, USA) under the ABI Prism 7500 Sequence Detector (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The reaction was run at one cycle of 50°C for 2 min and 95°C for 15 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. We used β-actin expression as an internal control. Specific primer sequences were synthesized in Biosune Biological Technology Corp (Shanghai, China), and the sequences of the primers are shown in Table 1 .
7
Quantitative Real-Time PCR for Gene Expression
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Total RNA was extracted using the RNeasy Mini kit (Qiagen) following the manufacturer’s protocol and then subjected to reverse transcription using the StarScript first strand cDNA synthesis kit (Transgen Biotech, Beijing, China). Real-time PCR was performed using SYBR Select Master Mix (Applied Biosystems) on an ABI PRISM 7500 Sequence Detector (Applied Biosystems). GAPDH served as an internal control for normalization [40 (link)].
The primers for RT-qPCR are listed as below:
S100A13 forward:
5’-TCCTAATGGCAGCAGAACCACTGA-3’ and
reverse:
5’- TTCTTCCTGATTTCCTTGGCCAGC-3’
IL-6 forward:
5’- TACCCCCAGGAGAAGATTCC -3’ and
reverse:
5’- TTTTCTGCCAGTGCCTCTTT -3’
IL-8 forward:
5’- TAGCAAAATTGAGGCCAAGG -3’ and
reverse:
5’- AAACCAAGGCACAGTGGAAC -3’
CXCL2 forward:
5’- GCCCAACGCACCGAATAGT-3’ and
reverse:
5’- CGCTGCCCATCATCATGAC-3’
CCL2 forward:
5’- AGTTCTTGCCGCCCTTCT -3’ and
reverse:
5’- GTGACTGGGGCATTGATTG -3’
IL-1β forward:
5’- GGGCCTCAAGGAAAAGAATC -3’ and
reverse:
5’- TTCTGCTTGAGAGGTGCTGA -3’
MMP3 forward:
5’- GCAGTTTGCTCAGCCTATCC -3’ and
reverse:
5’- GAGTGTCGGAGTCCAGCTTC -3’
IL-7 forward:
5’- CGCAAGTTGAGGCAATTTCT -3’ and
reverse: 5’- CTCTTTGTTGGTTGGGCTTC -3’
CXCL1 forward:
5’- AGGGAATTCACCCCAAGAAC -3’ and
reverse:
5’- TGGATTTGTCACTGTTCAGCA -3’
GAPDH forward:
5’- CGACCACTTTGTCAAGCTCA -3’ and
reverse:
5’- AGGGGTCTACATGGCAACTG -3’
The primers for RT-qPCR are listed as below:
S100A13 forward:
5’-TCCTAATGGCAGCAGAACCACTGA-3’ and
reverse:
5’- TTCTTCCTGATTTCCTTGGCCAGC-3’
IL-6 forward:
5’- TACCCCCAGGAGAAGATTCC -3’ and
reverse:
5’- TTTTCTGCCAGTGCCTCTTT -3’
IL-8 forward:
5’- TAGCAAAATTGAGGCCAAGG -3’ and
reverse:
5’- AAACCAAGGCACAGTGGAAC -3’
CXCL2 forward:
5’- GCCCAACGCACCGAATAGT-3’ and
reverse:
5’- CGCTGCCCATCATCATGAC-3’
CCL2 forward:
5’- AGTTCTTGCCGCCCTTCT -3’ and
reverse:
5’- GTGACTGGGGCATTGATTG -3’
IL-1β forward:
5’- GGGCCTCAAGGAAAAGAATC -3’ and
reverse:
5’- TTCTGCTTGAGAGGTGCTGA -3’
MMP3 forward:
5’- GCAGTTTGCTCAGCCTATCC -3’ and
reverse:
5’- GAGTGTCGGAGTCCAGCTTC -3’
IL-7 forward:
5’- CGCAAGTTGAGGCAATTTCT -3’ and
reverse: 5’- CTCTTTGTTGGTTGGGCTTC -3’
CXCL1 forward:
5’- AGGGAATTCACCCCAAGAAC -3’ and
reverse:
5’- TGGATTTGTCACTGTTCAGCA -3’
GAPDH forward:
5’- CGACCACTTTGTCAAGCTCA -3’ and
reverse:
5’- AGGGGTCTACATGGCAACTG -3’
8
Quantifying NorA Expression in S. aureus
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RNA extraction from the S. aureus strain was performed using the SV Total RNA Isolation System (Promega, Madison, WI, USA) based to the manufacturer’s indications. Complementary DNA (cDNA) was synthesized from total RNA by reverse transcription (RT) using the GoScript called Transcription System (Promega, Madison, WI, USA). NorA expression was assessed by qPCR, performed in triplicates using a Power SYBR® Green PCR Master Mix (Applied Biosystems Co., Foster City, CA, USA) and an ABI PRISM 7500 sequence detector (Applied Biosystems, Foster City, CA, USA). The amount of cDNA was used as a template in 20 μL reaction, along with 10 μL Power SYBR® Green and 1 pmol of each primer. The 16S gene was chosen to be used as an endogenous control. Table 1 shows the primers used in this study. The relative norA gene expression was determined using the ΔΔTc method.
9
Quantitative RT-PCR Analysis of Immune Markers
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Real-time PCR (QT-PCR) for CLM-1 transcript detection was performed using TaqMan gene expression assay for CLM-1. For the detection of CCL-17, COX-2, IL-1β, IL-6, TNFα, RELMα, NOS-2 transcripts, QT-PCR was carried out with the SYBR Green PCR Master Mix (Applied Biosystems). Primers were used at a final concentration of 200 nM (S1 Table ). TaqMan probe of eukaryotic 18s was used for cycle normalization (Applied Biosystems). Samples were run for 40 cycles (30 secs 95°C, 1 min 60°C, 30 secs 72°C) on an ABI- Prism 7500 Sequence Detector (Applied Biosystems). Data were calculated by the 2-Delta or the 2 (Delta-Delta CT) Pfaffl methods, using as a normalizer 18s. All PCR reactions were set up in triplicates.
10
Quantification of miRNA-221/222 and PTEN mRNA
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Total miRNA and mRNA were extracted using the miRNeasy mini kit (Qiagen GmbH, Hilden, Germany) and the RNeasy Micro kit (Qiagen GmbH) according to the manufacturer's protocol. RT-qPCR was performed using the Primescript RT reagent kit (Takara, Otsu, Japan) with U6 and 18S rRNA as the loading controls. For the detection of miR-221/222 and PTEN, the SYBR Green PCR assay (SYBR® Premix Ex Taq™; Takara) was performed and the primer sequences used were as follows: miR-221 forward, ACACTCCAGCTGGGAGCTACATTGTCTGCTGG and reverse, CTCAACTGGTGTCGTGGA; miR-222 forward, ACACTCCAGCTGGGAGCTACATCTGGCTACTG and reverse, CTCAACTGGTGTCGTGGA; U6 forward, CTCGCTTCGGCAGCACA and reverse, AACGCTTCACGAATTTGCGT; PTEN forward, TTGTGGTCTGCCAGCTAAA and reverse, CGCTCTATACTGCAAATGCT; 18S rRNA forward, CCTGGATACCGCAGCTAGGA and reverse, GCGGCGCAATACGAATGCCCC. The PCR was performed in triplicate consisting of 40 cycles of a denaturation step at 95°C for 5 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec after a cycle of a pre-denaturation step at 95°C for 30 sec on an ABI PRISM® 7500 Sequence Detector (Applied Biosystems, Foster City, CA, USA). Relative expression levels were calculated using the 2−ΔΔCq method (24 (link)).
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