The relative expression of the osteogenic genes Runt-related transcription factor 2 (RUNX2) and ALP and the adipogenic genes peroxisome proliferator-activated receptor gamma (PPARγ) and adipocyte protein 2 (AP2) (see Additional file 1 : Table 3 for primer sequences) was assessed as previously described [36 (link)]. Gene expression was performed with Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, Cheshire, UK) on an ABI Prism 7300 real-time PCR system (Thermo Fisher Scientific). The housekeeping gene ribosomal phosphoprotein P0 (RPLP0) was used as an internal control, and the relative expression of the studied genes was calculated using a mathematical model as previously described [37 (link)]. The expression levels of the inflammatory markers interleukin 6 (IL-6) and tumor necrosis factor (TNF) were analyzed with TaqMan Gene Expression Assays (Applied Biosystems) using an ABI Prism 7300 real-time PCR system (see Additional file 1 : Table 4). The results were calculated using the delta Ct method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control.
Abi prism 7300
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, United Kingdom, Italy, China, France, Canada, Belgium
The ABI Prism 7300 is a real-time PCR system designed for gene expression analysis and quantification. It features a 96-well thermal cycler, a sensitive optical detection system, and intuitive software for data analysis.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (71 mentions)
Trizol, by Thermo Fisher Scientific (32 mentions)
Rneasy mini kit, by Qiagen (29 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (23 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (18 mentions)
Primescript rt reagent kit, by Takara Bio (15 mentions)
Sybr premix ex taq, by Takara Bio (14 mentions)
Iscript cdna synthesis kit, by Bio-Rad (12 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (10 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (9 mentions)
Rneasy kit, by Qiagen (8 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (8 mentions)
Rneasy plus mini kit, by Qiagen (7 mentions)
Thunderbird sybr qpcr mix, by Toyobo (7 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (7 mentions)
Premix ex taq sybr green pcr, by Takara Bio (7 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (6 mentions)
Taqman primers and probe, by Thermo Fisher Scientific (6 mentions)
M mlv reverse transcriptase, by Promega (6 mentions)
380 protocols using abi prism 7300
1
Evaluating Osteogenic and Adipogenic Genes
2
Quantification of Pluripotency and Lineage Markers
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Total RNAs from cell samples were extracted using a miRNeasy Mini extraction kit (Qiagen) and the cDNA was reversely transcribed using a First-Strand Synthesis kit (Invitrogen). qRT-PCR analyses of pluripotent markers (Oct4, Sox2, NANOG, and KLF4), endoderm markers (FOXA2, AFP, and SOX17), mesoderm markers (MESP1, Brachyury, and Mixl1), ectoderm markers (SOX1, PAX6, and Notch1), CM specific markers during cardiovascular differentiation (NKX2.5, GATA4, cTnT, α-MHC, and β-MHC), and the transcription factor-serum response factor (SRF) were performed using gene specific primers (see supplementary Table 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/2524092 ) and Power SYBR Green PCR Master Mix in an ABI Prism 7300 (Applied Biosystems). Results were normalized to GAPDH transcripts and analyzed using 2−ΔΔCt method.
To quantify miR-1 expression in iPSCs treated with RSV, total RNAs were extracted using the mirVana PARIS Kit (Ambion, USA). Then, the miRNA specific cDNA was reversely transcribed using the TaqMan MicroRNA Reverse Transcription Kit. To quantify the expression of miR-1, TaqMan MicroRNA Assay Kit (Applied Biosystems) was used. U6 snRNA served as the internal control. qRT-PCR was performed in an ABI Prism 7300 (Applied Biosystems).
To quantify miR-1 expression in iPSCs treated with RSV, total RNAs were extracted using the mirVana PARIS Kit (Ambion, USA). Then, the miRNA specific cDNA was reversely transcribed using the TaqMan MicroRNA Reverse Transcription Kit. To quantify the expression of miR-1, TaqMan MicroRNA Assay Kit (Applied Biosystems) was used. U6 snRNA served as the internal control. qRT-PCR was performed in an ABI Prism 7300 (Applied Biosystems).
3
Evaluating Apoptosis Regulator Expression
4
Heat-Induced Expression Analysis of HSP18.2 Transgenic Strains
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Example 10
(10) Expression Analysis in Flower Stalks for the HSP18.2: iHinP1I Transgenic Strain and the HSP18.2: iMseI Transgenic Strain
From the plants used in the analysis in (9), flower buds and cauline leaves were each sampled from the plants immediately after carrying out the 3 hour/37 deg C. heat treatment. Total RNA extraction from each sample was performed using an RNeasy Plant Mini Kit (Qiagen), and a reverse transcription reaction was then run using a High-Capacity RNA-to-cDNA Kit (Life Technologies Corporation) to prepare cDNA. The expression of the 18SrRNA and BRCA1 genes was subsequently analyzed by real-time PCR (ABI PRISM 7300) using Power SYBR Green PCR Master Mix (Life Technologies Corporation). The results are given in
As shown in
5
Rice Plant RNA Expression Quantification
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Total RNA was extracted from various tissues of rice plants as well as cultured cells. Real-time RT-PCR assays were performed as described by Kurusu et al.49 (link) Real-time PCR was performed using an ABI PRISM 7300 sequence detection system (Life Technologies) with SYBR Green real-time PCR Master Mix (Toyobo, QPS-201) and the following OsATG7-specific primers: OsATG7-RealF, 5′-GATCGTGACA GCCCAAAAGC A-3′; and OsATG7-RealR, 5′-GACCAAGTGG ACCCTCACTG C-3′.50 (link) Relative mRNA levels were calculated using the standard curve method and normalized to corresponding OsEF1α (locus ID: Os03g0177500) gene levels.51 (link) Standard samples of known template amounts were used to quantify PCR products.
6
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from frozen liver specimens or cultured BMSCs with or without treatments, using an RNeasy kit (Qiagen, Hilden, Germany). Real-time RT-PCR was performed with an ABI Prism 7300 sequence-detecting system (Life Technologies, Foster City, CA), as described previously [15 (link)]. Primers (MWG Biotech, Ebersberg, Germany) used for real-time RT-PCR were as follows: 18 s rRNA, sense, 5′-GTA ACC CGT TGA ACC CCA TT-3′, and antisense, 5′-CCA TCC AAT CGG TAG TAG CG-3′; PPARγ: sense, 5′-GCC CAC CAA CTT CGG AAT C-3′, and antisense, 5′-TGC GAG TGG TCT TCC ATC AC-3′.
7
Quantifying Gene Expression in Zebrafish
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Total RNA was extracted from control or ccdc80-KO albino zebrafish at 6 dpf using an RNAqueous Micro Kit (Takara, Kyoto, Japan) according to the manufacturer’s protocol. RNA concentrations were determined using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and aliquots were reverse transcribed using an iScript Select cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). qPCR was performed using an ABI Prism 7300 (Life Technologies, Carlsbad, CA, USA) with SYBR Green Realtime PCR Master Mix Plus (Toyobo). The thermal cycling conditions were: 95°C for 1 min followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. We measured the expression of collagen type I alpha 1 (col1a1), secreted frizzled-related protein 2 (sfrp2), endothelin-1 (edn1), and glyceraldehyde-3-phosphate dehydrogenase (gapdh) mRNA. The data were normalized to gapdh mRNA levels to correct for variability in the initial template concentration and the conversion efficiency of the reverse transcription reaction. The primer sequences are shown in Supplementary Table S3 .
8
Quantification of fads2 Expression in Zebrafish
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Total RNA was extracted from zebrafish exposed to intense light
or normal light conditions in the presence or absence of fatostatin using an
RNAqueous Micro Kit (Takara, Kyoto, Japan) according to the manufacturer’s
protocol. RNA concentrations were determined using a NanoDrop spectrophotometer
(Thermo Scientific, Waltham, MA, USA), and cDNAs were generated using a ReverTra
Ace qPCR RT Kit (Toyobo). qPCR was performed using an ABI Prism 7300 (Life
Technologies Carlsbad, CA, USA) with THUNDERBIRD SYBR qPCR Mix (Toyobo). The
thermal cycling conditions were: 95 °C for 1 min followed by 40 cycles of 95 °C
for 15 s, 60 °C for 15 s, and 72 °C for 45 s. We measured the expression of
fads2 and eukaryotic translation elongation factor 1
alpha 1 like 1 (eef1a1l1) mRNA. The
fads2 mRNA levels were normalized to
eef1a1l1 mRNA levels to correct for variability in the
initial template concentration and the conversion efficiency of the reverse
transcription reaction. The primer sequences are shown in Table S25.
or normal light conditions in the presence or absence of fatostatin using an
RNAqueous Micro Kit (Takara, Kyoto, Japan) according to the manufacturer’s
protocol. RNA concentrations were determined using a NanoDrop spectrophotometer
(Thermo Scientific, Waltham, MA, USA), and cDNAs were generated using a ReverTra
Ace qPCR RT Kit (Toyobo). qPCR was performed using an ABI Prism 7300 (Life
Technologies Carlsbad, CA, USA) with THUNDERBIRD SYBR qPCR Mix (Toyobo). The
thermal cycling conditions were: 95 °C for 1 min followed by 40 cycles of 95 °C
for 15 s, 60 °C for 15 s, and 72 °C for 45 s. We measured the expression of
fads2 and eukaryotic translation elongation factor 1
alpha 1 like 1 (eef1a1l1) mRNA. The
fads2 mRNA levels were normalized to
eef1a1l1 mRNA levels to correct for variability in the
initial template concentration and the conversion efficiency of the reverse
transcription reaction. The primer sequences are shown in Table S25.
9
Quantification of Inflammatory Markers in Mice
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Total RNA of RAW264.7 cells was prepared using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany). Total mRNA was extracted from whole biopsy of the ear after sacrificing the mice using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Expression levels of IL-6, IL-1β, IL-17A and COX-2 mRNA were measured by real-time PCR using the ABI Prism 7300 sequence detection system (Life Technologies Corporation, Garlsbad, CA, USA) and normalized to GAPDH mRNA. The primers used were: 5′- CTGGAGCCCACCAAGAACGA-3′ (forward) and 5′- GCCTCCGACTTGTGAAGTGGT-3′(reverse) for IL-6 mRNA, 5′- TCCAGGATGAGGACATGAGCAC-3′ (forward) and 5′- GAACGTCACACACCAGCAGGTTA-3′ (reverse) for IL-1β mRNA, 5′- CAC CTC ACA CGA GGC ACA AG-3′ (forward), and 5′-GCA GCA ACA GCA TCA GAG ACA-3′ (reverse) for IL-17A, 5′-GTG TGC GAC ATA CTC AAG CAG GA-3′ (forward), and 5′-TGA AGT GGT AAC CGC TCA GGT G-3′ (reverse) for COX-2, 5′- AAATGGTGAAGGTCGGTGTG-3′ (forward) and 5′- TGAAGGGGTCGTTGATGG-3′(reverse) for GAPDH mRNA.
10
Quantitative Real-time PCR for NR4A1 and HSP90B1
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Expression of NR4A1 and HSP90B1 mRNA was quantitatively determined by real-time PCR on an ABI Prism 7300 sequence detection system (Life Technologies Corporation, Carlsbad, CA, USA). Total RNAs were prepared using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany). Expression level of the targeted mRNAs was normalized to β-actin mRNA. The primers used were: 5’-ATC TTG GGA TTC TCC CTT CG-3’ (sense) and 5’-TCC CAT ATT GGG CTT GGA TA-3’ (antisense) for NR4A1 mRNA, 5’-TGT AAT TGC TGA CCC AAG AGG-3’ (sense) and 5’-TCC AAT TCA AGG TAA TCA GAT GC-3’ (antisense) for HSP90B1 mRNA, and 5’-GCA CAG AGC CTC GCC TT-3’ (sense) and 5’-GTT GTC GAC GAC GAG CG-3’ (antisense) for ß-actin mRNA.
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