qRT-PCR was performed using the iTaq™ Universal SYBR Green Supermix (Bio-Rad Laboratories, CA, USA) on a 7500HT Real-Time PCR System (Life Technologies, USA). Real-time PCR primers used for GAPDH are (forward: 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse: 5′-TGTAGACCATGTAGTTGAGGTCA-3′); TRAP (forward: 5′-CACTCCCACCCTGAGATTTGT-3′, reverse: 5′-CATCGTCTGCACGGTTCTG-3′); CK (forward: 5′-GAAGAAGACTCACCAGAAGCAG-3′, reverse: 5′-TCCAGGTTATGGGCAGAGATT-3′); MMP-9 (forward: 5′-CTCAGAGATTCTCCGTGTCCTGTA-3′, reverse: 5′-GACTGCCAGGAAGACCTTGGTTA-3′).
7500ht real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7500HT Real-Time PCR System is a laboratory instrument designed for the amplification and detection of nucleic acid sequences. It utilizes real-time PCR technology to perform quantitative analysis of DNA, RNA, or cDNA targets. The system features a thermal cycler and a detector to monitor the fluorescence emitted during the PCR reaction in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Primescript rt reagent kit, by Takara Bio (5 mentions)
Fast sybr green pcr master mix, by Thermo Fisher Scientific (5 mentions)
Qscript cdna synthesis kit, by Quantabio (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Reverse transcription kit, by Takara Bio (3 mentions)
Sybr green pcr kit, by Takara Bio (3 mentions)
Gotaq dna polymerase, by Promega (2 mentions)
Illustra rnaspin mini rna isolation kit, by GE Healthcare (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Rnaspin mini rna isolation kit, by GE Healthcare (2 mentions)
Facsaria 2, by BD (2 mentions)
Power sybr green, by Takara Bio (2 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Prepseq residual dna sample preparation kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Quick rna miniprep kit, by Zymo Research (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
27 protocols using 7500ht real time pcr system
1
Quantitative Real-Time PCR Analysis of Osteoclast Markers
2
Quantification of Residual DNA
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Residual DNA in the samples was measured using real-time quantitative PCR (RT-qPCR). PrepSEQ residual DNA Sample Preparation Kit (Life Technologies) was used to prepare samples. RT-qPCR was carried out with the 2× TaqMan Universal PCR Master Mix Kit (Life Technologies) and 7500 HT Real-Time PCR system (Life Technologies) according to the manufacturer’s instructions.
3
RNA Isolation and qPCR Analysis of ICC-IM
4
Enriching Intestinal Interstitial Cells
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Jejunal ICC were dispersed from Kit+/copGFP mice as previously described (Zhu et al., 2009 (link)). Enriched populations of ICC were sorted by FACS (FACSAria II; Becton-Dickinson) using an excitation laser (488 nm) and emission filter (530/30 nm). Sorting was performed using a 130-μm nozzle and a sheath pressure of 12 psi. RNA was prepared from sorted ICC and dispersed unsorted jejunal cells of the tunica muscularis before sorting using an illustra RNAspin Mini RNA Isolation Kit (GE Healthcare). The PCR primers used and their GenBank accession numbers are listed in Table 1 . Quantitative PCR (qPCR) was performed using SYBR green chemistry on the 7500 HT Real-time PCR System (Applied Biosystems) and analyzed, as previously described (Baker et al., 2016 (link)).
5
Quantitative Real-Time PCR Protocol
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QRT-PCR was performed as previously described30 (link), 31 (link). Briefly, 2 μg of total RNA was reverse-transcribed into cDNA using a Reverse Transcription Kit (Takara, RR037A). QRT-PCR analyses were performed with Power SYBR Green (Takara, RR820A) in 7500HT Real-Time PCR System (Applied Biosystems, Foster City, CA). GAPDH internal control was used as an endogenous control, and fold changes were presented by using the 2-ΔΔCt method using the equation (ΔΔCT = (Ct gene of interest - Ct GAPDH) treated sample - (Ct gene of interest - Ct GAPDH) control sample). All qRT-PCR reactions were performed in triplicates. The fold change > 2 or < 0.5 was considered as significant.
6
Quantifying T-Helper Cell Subsets
7
Quantitative Real-Time PCR Assay
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Total RNA was extracted using TRIzol reagent (Invitrogen) and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). 200 ng RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China), and PCR amplifications were then carried out using a SYBR Green PCR Kit (TaKaRa) on a 7500HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Data were analyzed using 2−ΔΔCt method [8 (link)]. GAPDH or U6 was employed as an internal control.
8
Quantitative Real-Time PCR Assay
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According to the manufacturer’s instructions (see detail in Additional file 2 ). Total RNA was extracted from tissues or cells by using TRIzol reagent (Invitrogen, USA). For the RT-PCR assay, the reverse transcription was performed from RNA to cDNA and PCR analyses were performed by a PrimeScript™ RT-PCR Kit (Takara, Japan). Quantitative real-time PCR (qPCR) was determined by SYBR Green I mix (Toyobo, Japan) in triplicate based on an Applied Biosystems 7500HT Real-Time PCR System. The mRNA relative expression was normalized to reference genes GAPDH and/or U6. The reaction assays and primers used for qPCR were listed in Table S3 and Table S4 .
9
Isolation and Characterization of Jejunal ICC
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Jejunal ICC were dispersed from Kit+/copGFP mice as previously described (Zhu et al., 2009 (link); Zhu et al., 2011 (link)). ICC were sorted and purified by FACS (FACSAria II; Becton-Dickinson) using an excitation laser (488 nm) and emission filter (530/30 nm). Sorting was performed using a 130-μm nozzle and a sheath pressure of 12 psi.
RNA was prepared from sorted ICC and dispersed jejunal cells of the tunica muscularis before sorting using an illustra RNAspin Mini RNA Isolation kit (GE Healthcare). The PCR primers used and their GenBank accession numbers are provided inTable 1 . qPCR was performed using SYBR green chemistry on the 7500 HT Real-time PCR System (Applied Biosystems) and analyzed, as previously described (Baker et al., 2016 (link)). All datasets were normalized to the housekeeping gene Gapdh.
RNA was prepared from sorted ICC and dispersed jejunal cells of the tunica muscularis before sorting using an illustra RNAspin Mini RNA Isolation kit (GE Healthcare). The PCR primers used and their GenBank accession numbers are provided in
10
Gene Expression Analysis by qPCR
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Total RNA was isolated from cells using TRIzol Reagent (Invitrogen). Cytoplasmic and nuclear RNA were separated and extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). RNA was then reverse transcribed to cDNA using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). qPCR reactions were then carried out on a 7500HT Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.) using SYBR Green PCR Master Mix (Applied Biosystems). The relative expression of genes was calculated using the 2−ΔΔCt method [7 (link)], and GAPDH or U6 was used as an internal control for normalization. The primer sequences were listed in Table 1 .
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