Cells (3×106) were directly lysed with 1 ml TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was extracted using phenol chloroform method. The concentration and quality of RNA was measured using ultraviolet spectrophotometry (NanoDrop™ ND2000; Thermo Fisher Scientific, Inc.). Subsequently, cDNA was obtained by RT from 1 µg RNA and stored at −20°C. RT of mRNA was performed using TIANScript II cDNA First Strand Synthesis kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. SuperReal PreMix (SYBR-Green) kit (Tiangen Biotech Co., Ltd.) was used to detect mRNA expression, using GAPDH as an internal reference. The reaction system (20 µl) was composed of 10 µl SYBR Premix EXTaq, 0.5 µl forward primer (STAT3, 5′-GGAGGAGGCATTCGGAAAG-3′; β-actin, 5′-AACGGCTCCGGCATGTGCAA-3′), 0.5 µl reverse primer (STAT3, 5′-TCGTTGGTGTCACACAGAT-3′; β-actin, 5′-CTTCTGACCCATGCCCACCA-3′), 2 µl cDNA and 7 µl ddH2O. The following thermocycling conditions were used: Initial denaturation at 95°C for 5 min; 46 cycles of denaturation at 95°C for 20 sec and annealing at 55°C for 20 sec; and a final extension at 72°C for 30 sec (iQ5 system; Bio-Rad Laboratories, Inc.). The 2−ΔΔCq method was used to calculate the relative expression of target mRNA against GAPDH (16 (link)). Each sample was tested in triplicate.
Superreal premix sybr green kit
Manufactured by Tiangen Biotech
Sourced in China
The SuperReal PreMix (SYBR Green) kit is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR Green I dye, buffer, and other necessary components for performing real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Rnaprep pure cell bacteria kit, by Tiangen Biotech (3 mentions)
Beacon designer software v7, by Premier Biosoft (2 mentions)
Fastquant rt kit, by Tiangen Biotech (2 mentions)
C1000 touch thermal cycler, by Bio-Rad (2 mentions)
Iq5 system, by Bio-Rad (1 mentions)
Nanodrop nd 2000, by Thermo Fisher Scientific (1 mentions)
Tianscript 2 cdna first strand synthesis kit, by Tiangen Biotech (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Fastquant rt super mix, by Tiangen Biotech (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Fastquant rt super mix kit, by Tiangen Biotech (1 mentions)
Dnase 1, by Tiangen Biotech (1 mentions)
Superscript 2 reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Quant reverse transcriptase kit, by Tiangen Biotech (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Tiangen Biotech (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
12 protocols using superreal premix sybr green kit
1
Quantitative RT-PCR Analysis of STAT3 Expression
2
Comprehensive Rice Transcriptome Analysis Across Growth Stages
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Total RNA was extracted from different tissues (including the roots, stems and leaves of rice seedlings (2 weeks) in the vegetative growth period, and panicles of 0.5, 2, 4, 8, 16 and 22.5 cm in the reproductive growth period, and panicles of 1, 5, 10 and 25 days after fertilization) using the RNeasy Plant Mini Kit (cat. nos. 74903 and 74904) according to the manufacturer’s instructions. 2 μg of total RNA was reverse‐transcribed into first‐strand cDNA with FastQuant RT Super Mix (#KR108; Tiangen, Beijing, China). qRT‐PCR was performed with a C1000 TouchTM Thermal Cycler (#785BR05170, Bio‐Rad, Hercules, CA) and SuperReal PreMix (SYBR Green) kit (#FP204‐02; TIANGEN). OsActin1 was used as a reference gene. Three biological replicates were performed. The primers used here were listed in Table S10 .
3
Quantitative Gene Expression Analysis via qRT-PCR
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Total cDNA was synthesized from 1 µg wild type or other transformants' RNA using the PrimeScript RT Reagent kit with gDNA Eraser (Takara, Japan). Quantitative real-time PCR (qRT-PCR) was performed using a SuperReal PreMix (SYBR Green) kit (TIANGEN, China) with the synthesized cDNA as template. The PCR conditions were as follows: 95°C for 30 s for initial denaturation and 40 cycles each consisting of 95°C for 20 s, 58°C for 30 s, and 72°C for 20 s. For this experiment, an FTC-3000 Real-Time PCR System (Funglyn Biotech, Canada) was used according to the manufacturer's instructions. The qRT-PCR was carried out with primers Actin-F and Actin-R for amplifying the reference control actin gene. Expression levels of genes hex1 (Accession no. KF356403), cu/zn superoxide dismutase (sod) (Accession no. JX481779), and cytochrome c (Accession no. JX481780) from strains T28 and KO40 were tested using the following primer sets: hex1-F/hex1-R, cu/zn sod-F/cu/zn sod-R, and cytochrome c-F/cytochrome c-R (Table S1 ), respectively. The qRT-PCR assay was carried out in three biological replicates with every reaction in triplicate, and the gene expressions level was determined using the 2−ΔΔCT method as previously described [27] (link). The efficiency of primers is shown in Tables S2 and S3 .
4
Total RNA Extraction of Bacillus sp. N16-5
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For total RNA extraction, Bacillus sp. N16‐5 was cultured in Horikoshi‐I overnight. After 4–6 h of cultivation, total RNA was extracted using the HiPure Bacteria RNA Kit (Magen, Guangzhou, China). Reverse transcription was performed using HiScript III qRT SuperMix (with gDNase) (Vazyme, Nanjing, China). qPCR analysis was conducted using the SuperReal Premix SYBR green kit (Tiangen Biotech, Beijing, China) based on 16S rRNA as the reference and the housekeeping gene ispH as a positive control. Relative quantities were calculated using the 2−ΔΔCt method (Schmittgen & Livak, 2008 (link)). All experiments were performed in triplicate.
5
Quantitative gene expression analysis
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Total RNA was extracted from different tissues and reverse‐transcribed into first‐strand cDNA using FastQuant RT Super Mix Kit (#KR108; TIANGEN). qRT‐PCR was performed using SuperReal PreMix (SYBR Green) Kit (#FP204‐02; TIANGEN) by a C1000 Touch™ Thermal Cycler (#785BR05170, Bio‐Rad). OsUBQ5 was used as the internal control. Three biological replicates were performed. The primers used for qRT‐PCR were listed in Table S9 .
6
Transcriptional Analysis of C. glutamicum Induced by Methanol, Formaldehyde, and Formate
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C. glutamicum ATCC 13032 was cultivated in CGXII minimal media supplemented with 5 g/L glucose as a carbon source. Different inducers, methanol (5 g/L), formaldehyde (15 mg/L), or formate (5 g/L), were added prior to inoculation. The cultures were incubated at 30 °C and with shaking at 220 rpm. Cells were collected in the mid-exponential phase (OD600nm ≈ 3) and total RNAs were isolated using the RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech, China). After treated with DNase I (Tiangen Biotech, China), total RNA samples were used to synthesize cDNAs using random primers and Fast Quant RT Kit (Tiangen Biotech, China). The resultant cDNAs were used as templates for qPCR analysis. The total RNA samples were also used as templates for qPCR to confirm that genomic DNA contamination during total RNA extraction was minimal. Specific primers for qPCR were designed using Beacon Designer software v7.7 (PREMIER Biosoft International, USA) (Supplementary Table 3 ). qPCR was performed using the SuperReal Premix SYBR Green Kit (Tiangen Biotech, China) and Applied Biosystems® 7500 Real-Time PCR System (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.
7
Quantification of Inflammatory Mediators in Lung Tissue
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TRIzol reagent (Takara Biotechnology Co., Ltd, Dalian, China) was used to extract the total RNA from lung tissue according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Following the manufacturer’s instructions, the extracted RNA was reverse transcribed into cDNA using a Superscript II reverse transcription kit (Invitrogen, Carlsbad, CA, USA). The mRNA expression levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and NADPH p67phox were analyzed by RT-qPCR (SuperReal PreMix SYBR-Green kit, Tiangen Biotech, Co., Ltd, Beijing, China). The 2-ΔΔCt method was used to analyze the samples, and β-actin was used as a control. The primers used are presented in Table 1 . The procedures were as follows: denaturation at 94°C for 30 s, annealing at 56°C for 1 min and extension at 72°C for 1 min.
8
Quantifying Bcl-xl Expression by qPCR
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Total RNA was extracted using an RNeasy Mini kit, according to the manufacturer's instructions (TianGen Biotech Co., Ltd.). Total RNA (2 µl) was reverse transcribed in a 20-µl reaction system using a Quant Reverse Transcriptase kit (TianGen Biotech Co., Ltd.). qPCR was performed using a 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.) and a SuperReal PreMix (SYBR Green) kit (TianGen Biotech Co., Ltd.). Primers used for qPCR amplification were as follows: Bcl-xl, forward, 5′-CCTGAATGACCACCTAGAGCCTT-3′ and reverse, 5′-TCATGCCCGTCAGGAACCAG-3′; 18S rRNA, forward, 5′-GTAACCCGTTGAACCCCATT-3′ and reverse, 5′-CCATCC AATCGGTAGTAGCG-3′. The cycling conditions used were as follows: Pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 sec; annealing at 55°C for 20 sec; extension at 68°C for 35 sec. A total of 40 cycles were performed. The relative expression of Bcl-xl was normalized to 18S rRNA and was calculated using the 2−ΔΔCq method (12 (link)).
9
Evaluating Anthocyanin Biosynthesis Genes in Blueberry
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The expression of potential candidate genes of anthocyanin biosynthesis pathway in blueberry such as PAL, C4H, 4CL, CHS, CHI, F3H, DFR, ANS and UFGT were examined by RT-qPCR. Specific primers used for RT-qPCR reactions were listed in Table 6 according to the open reading frames of the target genes. Real-time qPCR reactions were carried out in a StepOnePlus Real-time PCR system (ABI, USA) using SuperRealPreMix (SYBR Green) kit (TIANGEN BIOTECH, Beijing, China) according to the manufacturer’s instructions. UBC28 gene was used as an internal control for normalization, and each sample was assayed in triplicate. Relative transcript levels were calculated and normalized as described previously [25 (link)].
10
Expression analysis of glycerol metabolism genes
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Cells of C. diolis DSM 15410 cultured using glycerol and mixed sugars (mass ratio = 1:1:1) as co-carbon sources were quickly collected in the middle of the exponential phase when OD620 was approximately 1.0. Cells incubated in pure glycerol medium were collected at the same time and taken as control. Total RNA was isolated using the RNAprep pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China) and then treated with DNase I (MBI, Fermentas, Lithuania). After that, cDNA was synthesized subsequently using random primes, SuperScript III Reverse Transcriptase (Invitrogen), and RNA as a template. Primes for dhaB1, dhaD, dhaT, and 16S rRNA were designed with Beacon Designer software. The primer sequences for dhaB1, dhaD, dhaT, and 16S rRNA were 5′-ACCTCAACCATCTCTATCAGTAAG-3′ (forward), and 5′-GGCTCAACACATCCAATTATTCC-3′ (reverse); 5′-CTTGGTGGTGGTAAAGCGATTG-3′ (forward), and 5′-TGGTGTGTAAAGAACTGCTGAATG-3′ (reverse); 5′-CTGTAGGTGGAGGGAGTTC-3′ (forward), 5′-AGTTAATACACAATGACGAGTTAC-3′ (reverse); 5′-GCGGAATACTTAATGCGTTAGC-3′ (forward), and 5′-TGCGTAGAGATTAGGAAGAATACC-3′ (reverse), respectively. Quantitative PCR analysis was then carried out by using SuperReal Premix SYBR Green kit (Tiangen Biotech, Beijing, China) and the CFX96 Real-time system (Bio-Rad) according to the manufacturers’ instructions.
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