Extraction and bisulfite conversion of genomic DNA from 230 primary HNSCC and 36 noncancerous mucosal samples were performed using the MethylEasy Xceed Rapid DNA Bisulfite Modification Kit (TaKaRa, Tokyo, Japan) per the manufacturer’s instructions [12 (link)]. The methylation levels of the CpG islands in the promoters of the SST, TAC1, HCRT, NPY, and GAL genes were determined via Q-MSP with the TaKaRa Thermal Cycler Dice Real Time System TP800 (TaKaRa); the primer sets are listed in Additional file 1 : Table S1. Exon structure and CpG sites within expanded views of the promoter region relative to the transcription start site (TSS) are presented in Additional file 2 : Figure S1. A standard curve was constructed by plotting known concentrations of serially diluted EpiScope Methylated HeLa gDNA (TaKaRa). The normalized methylation value (NMV) was determined as follows: NMV = (Target gene-S/Target gene-FM)/(ACTB-S/ACTB-FM), where Target gene-S and Target gene-FM represent the target gene methylation levels in the tumor sample and universal methylated DNA control, respectively, and ACTB-S and ACTB-FM represent the ACTB (which encodes β-actin) methylation levels in the sample and control, respectively. Analysis was performed using the software (version 1.03A) for the Thermal Cycler Dice Real Time System TP800 (TaKaRa), according to the manufacturer’s directions [13 (link)].
Thermal cycler dice real time system tp800
Manufactured by Takara Bio
Sourced in Japan, United States, China
The Thermal Cycler Dice Real Time System TP800 is a laboratory instrument designed for real-time PCR (polymerase chain reaction) applications. It is capable of performing precise temperature cycling for DNA amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (23 mentions)
Sybr premix ex taq, by Takara Bio (22 mentions)
Sybr premix ex taq 2, by Takara Bio (22 mentions)
Primescript rt reagent kit, by Takara Bio (18 mentions)
Primescript rt master mix, by Takara Bio (14 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
Rneasy plus mini kit, by Qiagen (7 mentions)
Thunderbird sybr qpcr mix, by Toyobo (6 mentions)
Thermal cycler dice real time system software, by Takara Bio (6 mentions)
Rnaiso plus, by Takara Bio (6 mentions)
Oligo dt primer, by Qiagen (5 mentions)
High pure rna isolation kit, by Roche (5 mentions)
Revertra ace, by Toyobo (5 mentions)
Sybr premix ex taq 2 tli rnaseh plus, by Takara Bio (5 mentions)
Quantitect reverse transcription kit, by Qiagen (5 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (4 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (4 mentions)
Rnaiso, by Takara Bio (4 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (4 mentions)
167 protocols using thermal cycler dice real time system tp800
1
Methylation Analysis of HNSCC Samples
2
Quantitative Gene Expression Analysis
3
qRT-PCR for Gene Expression Analysis
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For cDNA synthesis, 500 ng of total RNA was reverse transcribed using the PrimeScript RT reagent Kit (Takara Corp., Otsu, Japan). cDNA equivalent to 5 ng of total RNA was used for all the PCR reactions. All the PCR reactions were performed using SYBR Premix Ex Taq (Takara Corp.) in a Thermal Cycler Dice Real Time System TP800 (TaKaRa). qRT-PCR was performed in duplicate for each sample using default two-step amplification procedures in accordance with the manufacturer's instructions. The standard curve method was used to determine the relative quantity of mRNA. All qRT-PCR data were normalized to GAPDH expression. PCR primers (PCReady primer) were purchased from Operon Biotechnology (Tokyo, Japan) and are described in Table S2.
4
Quantitative Real-Time PCR for Gene Expression
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TAs were prepared and mechanistically homogenized using Bullet Blender Storm (Next Advance, NY), and total RNA was extracted from homogenized TA or cultured cells with RNAiso (Takara, Kusatsu, Japan) following the manufacturer’s instructions. One microgram of RNA was converted to cDNA using oligo dT primers (Qiagen, Hilden, Germany) and Reverse Transcriptase XL (AMV) (Takara, Kusatsu, Japan). Gene expression was assessed using quantitative real-time PCR with Thermal Cycler Dice Real Time System TP800 (Takara, Kusatsu, Japan) and SYBR Premix Ex Taq (Takara, Kusatsu, Japan). Primers used in this study are listed in Supplementary Table S1A .
5
Quantitative Real-Time PCR Analysis of PalbHLH1 and PalMYB90
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Quantitative real-time PCR (qRT-PCR) analysis was performed using a Thermal Cycler Dice Real-Time System TP800 (TaKaRa, Dalian, China) and the specific primers for PalbHLH1 and PalMYB90 shown in Table S3 Datasheet 1 Table S1 Datasheet 1
6
Quantitative Gene Expression Analysis
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Total RNA was extracted using the RNAiso reagent (TaKaRa, Shiga, Japan), and reverse transcribed into cDNA with a PrimeScript Double Strand cDNA Synthesis Kit (TaKaRa), following the manufacturer’s instructions. Gene expression levels were quantified by quantitative real-time PCR (qRT-PCR) using SYBR Premix ExTaq II (TaKaRa) with a Thermal Cycler Dice Real-Time System TP800 (TaKaRa). Transcript abundances were calculated according to the comparative CT method [19 (link)]. For each sample, at least three biological replicates were analyzed, and each with three repeats. ACT2 was used as a reference for normalizing gene expression. All primers used in this study are listed in Supplemental Table S2 .
7
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was extracted from kidney samples using the NucleoSpin® TriPrep Kit (Takara Bio Inc.) and reverse transcribed into cDNA using PrimeScriptTM RT Master Mix (Perfect Real Time; Takara Bio Inc.). The gene segments were then amplified from the synthesized cDNA using the Thermal Cycler Dice Real Time System TP800 (Takara Bio Inc.), SYBR® Premix Ex Taq™ (Tli RNaseH Plus; Takara Bio Inc), and specific gene primers (Supplementary Tables S4 and S5 ). The 18S ribosomal RNA (18S rRNA) and beta-actin (Actb) genes were used as internal controls. Each sample was tested in duplicate for the average Ct value. Actb or 18S rRNA was used to calculate the mean normalized expression values of the target gene transcripts.
8
Quantitative Real-Time PCR Analysis Protocol
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Total RNA isolation and quantitative real-time PCR analysis were performed as described previously56 (link)57 (link). Briefly, for real-time PCR, performed with a Thermal Cycler Dice Real Time System TP800 (Takara, Kyoto, Japan), cDNAs were diluted 1:25 in the reaction mixture consisting of SYBR Premix EX Taq II (Takara). The real-time PCR program consisted of hot start enzyme activation at 95 °C for 10 s, 40 cycles of amplification at 95 °C for 10 s and 60 °C for 40 s. Finally, to obtain the dissociation curve, a final cycle was performed at 95 °C for 1 min, 60 °C for 30 s, and then 95 °C for 10 s. For data analysis, mouse β-actin (Actb) or glyceraldehyde-3-phosphate dehydrogenase (Gapdh) housekeeping genes was used as internal control. Induction values were calculated using analysis software. Primer sequences are available upon request from the Takara Bio Inc. website (http://www.takara-bio.co.jp/).
9
Quantifying Gut Microbial Abundance
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The universal 16S rRNA primers (27Fmod: 5′-AGRGTTTGATYMTGGCTCAG-3′ and 338R: 5′-TGCTGCCTCCCGTAGGAGT-3′) were used to estimate the microbial cell number by real-time qPCR. DNA of Escherichia coli that had been counted by the Colony Forming Unit (CFU) assay was used as a standard. Fecal DNA was assayed in 20 μL PCR reactions according to the protocol for TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa Bio, Tokyo, Japan). Each reaction mixture contained 10 pmol of each primer, 10 µL of TB Green Premix Ex Taq II (Tli RNase H Plus) (Takara Bio, Tokyo, Japan), extracted DNA, and UltraPure DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, Waltham, MA, USA) to reach a final volume of 20 μL. PCR conditions were as follows: 95 °C for 30 s and then 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The threshold cycle (Ct) value of qPCR was calculated by the 2nd Derivative Maximum (SDM) method using the Thermal Cycler Dice Real Time System TP800 (Takara Bio, Tokyo, Japan). To estimate the absolute abundance of bacteria in the fecal sample, the log10-fold standard curves ranging from 104 to 109 copies were produced using the DNA of E. coli. The threshold cycle values were converted into the estimates of the absolute abundance of bacteria in the fecal samples (copy numbers/g of dry feces).
10
Quantification of VEGF mRNA Expression
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Total RNA from tissues was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed to cDNA by using a PrimeScript RT Master Mix Kit (Takara Bio Inc., Otsu, Japan). cDNA was generated using a Thermal Cycler Dice Real Time System TP800 (Takara Bio Inc.) with qPCR SYBR Green to determine the transcriptional expression of specific genes. The qPCR condition was amplified as follows: 50 cycles of denaturation at 9 °C 5 s, annealing at 6 °C 30 s. Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Relative gene expression was calculated by the 2ddCt method. The primer sequences were as follows: glyceraldehyde-3-phosphate dehydrogenase, (F): 5′-AACAGCAACTCCCACTCTTC-3′ and (R): 5′-CCTGTTGCTGTAGCCGTATT-3′; and VEGF, (F): 5′-AGGCTGCTGTAACGATGAAG-3′ and (R): 5′-TCTCCTATGTGCTGGCTTTG-3′.
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