Thunderbird sybr qpcr kit

Allele specific qPCR was performed on cDNA prepared from sorted cells or neocortical tissues of F1 hybrid mice obtained by crossing BL6 and JF1. For other primer sets, qPCR was carried out with the use of cDNA prepared from neocortical tissues of BL6 mice. Total RNA was extracted using RNAiso Plus (Takara) following the instructions of the manufacturer. Reverse transcription (RT) was performed with at the maximum of 0.5 μg of total RNA and ReverTra Ace qPCR Master Mix with gDNA remover (TOYOBO). The obtained cDNA was subjected to real-time PCR analysis in a Roche LightCycler 480 II with THUNDERBIRD SYBR qPCR kit (TOYOBO). As for allele specific qPCR, plasmid DNA containing each SNPs was used as a standard and relative copy number was calculated according to the molecular weight. In other experiments, the amount of mRNA quantified was normalized relative to that of β-actin mRNA.
The used primers were as follows:

Total RNA was extracted from cultured NCI-H292 cells after 48 h treatment with dopamine (1 μM), A68930 (1 μM), isoproterenol (1 μM), or CSE (10%) using the RNeasy Mini Kit (QIAGEN, Valencia, CA). Total RNA was transcribed into cDNA using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) in accordance with the manufacturer’s instructions. Quantitative real-time PCR on the CFX96 Real-Time PCR Detection System (Bio-Rad) was performed using Thunderbird SYBR qPCR kit (Toyobo) according to manufacturer’s instruction. Primer sequences for MUC5AC and GAPDH were shown in Table 1. The specificity of amplification was confirmed by melting curve analysis. The Ct value determined by the CFX manager Software (Bio-Rad) for all samples was normalized to the housekeeping gene GAPDH, and the relative fold induction against untreated controls was computed by the comparative Ct (ΔΔCt) method.Table 1

Primer sequences

Target	Sequence of Primer	Amplicon size (bp)
Human MUC5AC	FP: 5′- GGA GGA AGC TGG CCC TGC TCT GG-3’	116
	RP: 5′- AGA GAG GGC AGG GTG GTG CTT GT-3’
Human GAPDH	FP: 5′- CCA GGG CTG CTT TTA ACT CTG GTA AAG TGG ATA-3’	173
	RP: 5′- CAT CGC CCC ACT TGA TTT TGG AGG GA −3’

FP forward primer, RP reverse primer

Total RNA was isolated from cells after 24 h of treatment, and the RNA content was measured. Reserve transcription was performed followed by ReverseTra Ace qPRC RT Kit (Toyobo). The gene-specific RT-PCR, targeting AGR2 and GAPDH, was conducted with ThunderBird SYBR qPCR Kit (Toyobo) and Applied Biosystems Real-Time PCR Instrument (Life Technology). The sequences of the designed mutagenesis primers are shown in Table 2. The mRNA fold changes were calculated according to the ΔΔCt value.Table 2

Primers sequence of RT-PCR assay

RT-PCR target	Primer sequence
AGR2	Antisense: 5′ GGAGGACAAACTGCTCTGCCAA 3′ Sense: 5′ TCCAAGACAACAAACCCTTG 3′
BCAR1	Antisense: 5′ CTGGAAAAGGGCAGCATCAC 3′ Sense: 5′ CTATGGGCCGTGACACCTC 3′
BCAR2	Antisense: 5′ CTATGGTCATGCCCGTGTCTG 3′ Sense: 5′ TTCCTGGGGATTTCGAGCAC 3′
GAPDH	Antisense: 5′ TGATGGCATGGACTGTGGTCATGAG 3′ Sense: 5′ CTCCTGCACCACCAACTGCTTAGC 3′

The primers sequences in the above table are used for RT-PCR process for detecting the mRNA level of target gene

Quantitative PCR was performed using the CFX96 thermal cycler (Bio-Rad, Hercules, CA) under the following program: 15 min at 95°C, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C. For plasma samples, qPCR was performed using the Thunderbird SYBR qPCR kit (Toyobo, Tokyo, Japan) in combination with the miScript Primer Assay for let-7g, miR-19b, miR-29b, miR-30a, miR-30d, miR-103, miR-126-5p, miR-144, miR-148a, miR-150, miR-155, miR-221, miR-223, miR-320a, miR-361, miR-425-5p, miR-451, miR-486, miR-489, miR-1249, and miR-2888 (Qiagen) according to the manufacturer’s protocol. For muscle samples, qPCR was performed using the Quantitect SYBR Green PCR Kit (Qiagen) in combination with RPL7 PCR primers [16 ]. Differences in the expression ratios of the target miRNA/let-7g for plasma samples and of the target miRNA/RPL7 for muscle samples were compared between the grazing periods as well as between the cattle groups [16 ]. Melting curve analysis was used to confirm the specificity of the amplification reactions.

Total RNA from tissues or cells was extracted using Trizol reagent and reverse‐transcribed into cDNA using High‐Capacity cDNA reverse transcription Kit [for messenger RNA (mRNA); Thermo Fisher Scientific] or TaqMan™ MicroRNA Reverse Transcription Kit (for miRNA; Thermo Fisher Scientific) following the manufacturer’s instructions. Quantitative RT‐PCR was performed using Thunderbird SYBR qPCR Kit (TOYOBO, Osaka, Japan) on the StepOnePlus Real‐Time PCR system (Applied Biosystems, Foster City, CA, USA). GAPDH and U6 small nuclear RNA were used as internal references for mRNA and miRNA, respectively. Primers were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The relative quantification in gene expression was determined using the 2^−ΔΔCt method [26].