Transcription expression level of target genes related to osteoblastic differentiation was detected with RT-qPCR. Total RNA of MC3T3-E1 cells exposed to A1254 (0, 2.5, 5, 10, and 20 μmol/L) for 12h was extracted with TRIzol reagent (Invitrogen, California, USA). Then, reverse transcription of RNA samples was carried out using a qPCR RT Kit (18091050, Invitrogen, California, USA). Subsequently, RT-qPCR was performed with SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) following the provided protocol. The amplification procedure was started at 95°C for 60 s, then followed by 50 cycles of denaturation at 95°C (15 s), annealing at 63°C (15 s), and extension at 72°C (45 s). RT-qPCR amplification reaction was conducted on a Bio-Rad iQ5 Real Time PCR (Bio-Rad, California, USA). The 2-ΔΔCt method was applied to calculate the fold changes of gene expression with Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as the calibrator. The primer sequences of bsp, ocn, trpv6, alp, and Gapdh were presented in Table 1
Iq5 real time pcr
Manufactured by Bio-Rad
Sourced in United States
The IQ5 real-time PCR is a laboratory instrument used for conducting quantitative real-time polymerase chain reaction (qPCR) experiments. It is capable of detecting and quantifying specific DNA or RNA sequences in a sample.
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Lab products found in correlation
Sybr premix ex taq, by Takara Bio (4 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (2 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Rnaeasy mini kit, by Qiagen (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Sybr green supermix, by Thermo Fisher Scientific (2 mentions)
Sybr green mix, by Bio-Rad (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Sybr green pcr master mix, by Toyobo (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rt qpcr kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cellamp direct rna prep kit, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Snord61 miscript primer assay, by Qiagen (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Miscript primer assay kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
23 protocols using iq5 real time pcr
1
Osteoblastic Differentiation Gene Expression
2
RT-qPCR Protocol for Gene Expression
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After culturing for 12 hours, the total RNA was extracted with TRIzol (Invitrogen) reagent and its quantity and integrity were evaluated with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA samples were reverse-transcribed using PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa Code: D6110A, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) according to the manufacturer's instructions. The real-time PCR was performed with a Bio-Rad IQ5 Real-Time PCR (Bio-Rad Laboratories, Inc., Hercules, CA, USA), using SYBR Premix Ex Taq II Kit (TaKaRa Code: DRR081A, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). The protocol in detail was described in our previous works [18 (link)]. Primers for target and internal reference gene (β-actin) were designed with Primer 5.0 and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (China). The information of primers was displayed in Table 1 .
3
Real-Time PCR Quantification of Gene Expression
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RNA samples were reverse-transcribed using PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa Code: D6110A, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China), according to the manufacturer’s instructions. The real time-PCR was performed in a Bio-Rad IQ5 Real-Time PCR (Bio-Rad Laboratories, Inc. Hercules, CA, US), using SYBR Premix Ex Taq II kit (TaKaRa Code: DRR081A, TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). All reactions were run using the protocol below: 30 s at 95°C; 10 s at 95°C, 20 s at annealing temperature, and 30 s at 72°C for 40 cycles. The same conditions were performed on an equal amount of RNAase-free water as a negative control. The information of primers for target and internal reference gene were displayed in table 1 including the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control gene. It is recognized that using a single ICG is not ideal [18] (link) but GAPDH has been used previously as the sole ICG in studies of mammary cell gene expression.
4
Quantitative Expression Analysis of Metabolic Genes
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The expression levels of gapN, gapC, alsS, ilvC, ilvD, kivd and yqhd were determined by RT-PCR, according to references [45 (link), 53 (link)]. The recombinant strains were cultured in M9 medium supplemented with 36 g/L glucose, 5 g/L yeast extract, and 1,000th dilution of Trace mix A5. Cells were harvested when OD600 reached 4.0. Total mRNA was extracted using the CellAmp® Direct RNA Prep Kit (Takara, Dalian, China) as described by the manufacturer. The cDNA was amplified using PrimeScript™ RT reagent Kit (Takara, Dalian, China) with the total mRNA as the templates. Samples were then analyzed using Bio-Rad iQ5 Real Time PCR (Bio-Rad, USA) with SYBR® Premix Ex Taq™ (Takara, Dalian, China). RT-PCR amplification primers are given in Additional file 1 : Table S1. The 16SrRNA gene was selected as internal standard. The obtained data were analyzed by using the 2−ΔΔCt method described previously [45 (link)].
5
Mature miRNA Profiling in Heart Tissue
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Mature miRs were analyzed by miScript II RT kit (Qiagen) and miScript SYBR Green PCR kit with miScript Primer Assay kit (Qiagen) according to manufacturer’s instructions. Briefly, RNA was isolated from left ventricles and cDNA for mature miR profiling was prepared using the miScript II RT kit. Mature miRa were determined by real-time PCR using the miScript SYBR Green PCR kit (Qiagen). cDNA template was diluted to 1 ng/μl in RNase free water. Two nanograms of template cDNA were used for miRs quantification in a final volume of 25 μl system containing specific primers and QuantiTect SYBR Green PCR master mix following manufacturer’s instructions. Primers included miScript Universal Primer, rat-specific miScript mature miRNA primers and SNORD61 miScript Primer Assay (Qiagen). Serial dilutions of the positive control were done on each plate to create a standard curve for the quantification. The real-time PCR was performed in triplicate and threshold cycle numbers were averaged for each sample using IQ5 real-time PCR (BioRad). The expression levels of each mature miR in control and losartan-treated heart tissues were computed following the method described by Livak and Schmittgen,[27 (link)] and expressed as fold of SNORD61.
6
Quantifying Tissue-Specific Expression of Smads in Buffalo
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In this study, according to the obtained CDSs of buffalo Smads 1, 4, and 5, three pairs of primers were designed to detect their tissue differential expression. The relative expression levels of Smads 1, 4, and 5, in ten tissues, were assayed by RT-qPCR using SYBR Premix Ex Taq (Takara, Dalian, China) and conducted on iQ5 Real-Time PCR (Bio-Rad, Hercules, CA, USA) followed the manufacturer’s instructions. Each reaction mixture contained 2 μL cDNA, and 0.5 μL or 10 μM for forward and reverse primers, respectively, 7 μL double-distilled water, and 10 μL SYBR Premix Ex Taq. The qPCR was executed initially at 95 °C for 30 s, then followed by 35 cycles with each cycle at 95 °C for 5 s, at 60 °C for 20 s and at 72 °C for 30 s. The β-actin (ACTB; NM_001290932), ribosomal protein S15 (RPS15; XM_006050525) and ribosomal protein S23 (RPS23; XM_006059350) were used as endogenous references for normalization of the Smads 1, 4, and 5 expression levels Table 1 . The qPCR data were analyzed using the method of −2 . The Ct = Ct − Ct . Ct = Ct − Ct . The geometric mean of the Ct values of the three reference genes obtained in this study was used to normalize the targeted mRNA expression.
7
Quantification of Cell Cycle Regulators
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RNA from the treated and untreated MCF-7 cells was extracted using the RNeasy Plus Mini Kit (Qiagen NV, Venlo, the Netherlands) according to the manufacturer’s protocol. The RNA quality was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA was converted to complementary (c)DNA using an iScript™ cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Expression of p21, PLK1, and FOXM1 were evaluated by qRT-PCR with an SYBR® Select Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on an iQ-5 Real Time PCR (Bio-Rad Laboratories Inc.) using the primers as stated in Table 1 and the following PCR conditions: one cycle of 50°C for 2 minutes for UDG (uracil-DNA glycosylase) activation; one cycle of 95°C for 2 minutes for DNA polymerase activation; 40 cycles of 95°C for 2 seconds for denature; and 60°C for 30 seconds for annealing and extension. Standard curves for housekeeping genes (β-actin, 18srRNA, and GAPDH) and target genes (p21, PLK1, and FOXM1) were generated from the amplification of the serially diluted RNA samples isolated from the control MCF-7 cell line to optimize the assay. Each of the, treated and untreated samples were run in triplicate and non-template control was included. Normalization of reference genes was done using geNorm.13 (link)
8
RNA Isolation and Real-Time PCR Analysis
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RNA isolation was performed using an RNAeasy Mini Kit (Qiagen) according to manufacturer’s recommendations. Reverse transcription (RT) was performed using SuperScript III First-Strand Synthesis System (Invitrogen). Real-time PCR was performed with Biorad iQ5 real-time PCR. Primers used are as follow: GAPDH-F 5′-CCACTTGAAGGGTGGAGCCA-3′; GAPDH-R 5′-TCATGGATGACCTTGGCCAG-3′; p21-F 5′-GCCCGAGAACGGTGGAACTT-3′; p21-R 5′GACAAGGCCACGTGGTCCTC-3′.
9
Tissue-Specific Expression Analysis of PPARGC1A
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To analyze tissue differential expression, a pair of primers were designed
according to the obtained CDS of buffalo PPARGC1A in this work. The
relative expression of PPARGC1A in 10 tissues during lactation and non-lactation
were assayed by qPCR fluorescent technology using SYBR Premix Ex Taq
(Takara) and performed on iQ5 Real Time PCR (Bio-Rad, USA) according to the
manufacturers' instructions. The 20 L reaction system included 2 L cDNA, 10 L SYBR Premix Ex Taq, 0.5 L of 10 M forward
primer, 0.5 L of 10 M reverse primer, and 7 L sterile water.
The qPCR amplification was carried out firstly at 95 C for 30 s,
then followed by 40 cycles of 95 C for 5 s, 60 C for
20 s and 72 C for 30 s. The -actin (ACTB; accession no.
NM_001290932) was used as an endogenous reference for
normalization of PPARGC1A expression profiles (Table 1). The data of qPCR were
analyzed through the 2 method, where and . All the treatments were replicated three
times. Significance of PPARGC1A mRNA level in multiple tissues between two periods
was determined via Student's test, which was established at a .
according to the obtained CDS of buffalo PPARGC1A in this work. The
relative expression of PPARGC1A in 10 tissues during lactation and non-lactation
were assayed by qPCR fluorescent technology using SYBR Premix Ex Taq
(Takara) and performed on iQ5 Real Time PCR (Bio-Rad, USA) according to the
manufacturers' instructions. The 20 L reaction system included 2 L cDNA, 10 L SYBR Premix Ex Taq, 0.5 L of 10 M forward
primer, 0.5 L of 10 M reverse primer, and 7 L sterile water.
The qPCR amplification was carried out firstly at 95 C for 30 s,
then followed by 40 cycles of 95 C for 5 s, 60 C for
20 s and 72 C for 30 s. The -actin (ACTB; accession no.
NM_001290932) was used as an endogenous reference for
normalization of PPARGC1A expression profiles (Table 1). The data of qPCR were
analyzed through the 2 method, where and . All the treatments were replicated three
times. Significance of PPARGC1A mRNA level in multiple tissues between two periods
was determined via Student's test, which was established at a .
10
RNA Isolation and Real-Time PCR Analysis
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