The collected cells and tissues were added to TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and total RNA was extracted according to the RNAiso Plus kit instructions (Takara Bio, Inc., Otsu, Japan). The RNA concentration was assessed using the NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). The FastQuant cDNA First-Strand Synthesis kit (Tiangen Biotech, Co., Ltd., Beijing, China) was used for reverse transcription. A qRT-PCR kit FastStart Universal SYBR Green Master Mix (Rox) (Roche Diagnostics GmbH, Mannheim, Germany) was used to produce the reaction system and amplification according to the manufacturer's instructions. The reaction cycling conditions were performed as follows: 1 cycle at 95°C for 15 min, followed by 40 cycles at 95°C for 10 sec and at 60°C for 60 sec, and then relative mRNA expression was analyzed using the comparative quantification cycle (Cq) method followed by normalization to β-actin expression (Applied Biosystems; Thermo Fisher Scientific, Inc.) (15 (link)). The primers used for amplification were as follows: PEA15 forward, 5′-GTTCTGTAGTCAACCACCAT-3′ and reverse, 5′-ACCAACAACATCACCCTT-3′; β-actin forward, 5′-TCTTCATGGTGCTGGGAG-3′) and reverse, 5′-AATGAGCGGTTCCGTTGC-3′ which was used as the internal control.
Fastquant cdna first strand synthesis kit
Manufactured by Tiangen Biotech
Sourced in China
The FastQuant cDNA first strand synthesis kit is a laboratory tool used for the reverse transcription of RNA into complementary DNA (cDNA). This kit provides the necessary reagents and enzymes to efficiently convert RNA into single-stranded cDNA, which can then be utilized for downstream applications such as gene expression analysis, PCR, and other molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Tgem micro spectrophotometer, by Tiangen Biotech (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rnaiso plus kit, by Takara Bio (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master mix rox, by Roche (1 mentions)
Total rna extraction reagent, by Takara Bio (1 mentions)
Rnaprep pure animal tissue total rna extraction kit, by Tiangen Biotech (1 mentions)
Kapa sybr fast qpcr kit, by Roche (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Taq pcr mastermix, by Tiangen Biotech (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Superreal premix plus, by Tiangen Biotech (1 mentions)
Animal tissue total rna extraction kit, by Tiangen Biotech (1 mentions)
Prism 8, by GraphPad (1 mentions)
Plant genomic dna kit, by Tiangen Biotech (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Lightcycler 96 real time system, by Roche (1 mentions)
19 protocols using fastquant cdna first strand synthesis kit
1
RNA Extraction and qRT-PCR for Gene Expression Analysis
2
Transcriptome Profiling of Plant Leaves and Roots
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Total RNA was extracted from leaves and roots of three group seedlings using TRIzol® Reagent (OmniPlant RNA Kit(DNase I))according to the manufacturer’s instructions (Invitrogen). The first-strand cDNA was synthesized with FastQuant cDNA First-Strand Synthesis Kit (Tiangen Biotechnology, Beijing). After adjusting the cDNA template concentration, qRT-PCR assays were detected by Fluorescence Quantification PCR Kit (Tiangen Biotechnology, Beijing) on StepOnePlusTM Real-Time PCR System (ABI, USA). The primer sequences of 20 DEGs were shown in Additional file 9 and VcUBC28 was used as the reference gene for analyzing expression data (Vashisth et al., 2011; Liang et al., 2019) [7 (link), 90 (link)]. The RNA-seq data were displayed by log10 (FPKM + 1). All the qRT- PCR experiments were performed for three biological replicates.
3
Total RNA Extraction and Reverse Transcription
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Following the addition of total RNA extraction reagent (Grand Island, NY, USA), total RNA was extracted from the collected cells using RNAiso™ Plus kits (Takara, Japan) according to the manufacturer’s instructions. RNA concentration was determined using a spectrophotometer (Beckman Coulter, USA). Reverse transcription was performed using the FastQuant cDNA first strand synthesis kit (TIANGEN, China) and amplified with HSF1-specific primers (Invitrogen, USA) using the TaKaRa RNA PCR kit (AMV, Ver. 3.0) according to the manufacturer’s instructions. The amplified products were analyzed by 1.5% agarose gel electrophoresis.
4
qRT-PCR Analysis of VDBP Expression
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Total RNA was isolated from tissue samples or cultured cells using the RNAprep Pure Animal Tissue Total RNA Extraction Kit (Tiangen Biotech, Beijing, China) or the Trizol reagent (Thermo Fisher Scientific), respectively. A Fast Quant cDNA first strand synthesis kit (Tiangen Biotech) was used to create the cDNA first strand. RT-PCR experiment was conducted in triplicate using the KAPA SYBR FAST qPCR Kit (KAPA Biosystems, Wilmington, DE, USA). Primer sequences were: 5′-GCTGACCCTGACTGCTGCTATGAC-3′ (forward) and 5′-CATGCAGAGCTTTCGGTTCC-3′ (reverse) for human VDBP; 5′-AGCCTCAAGATCATCAGCAATGCC-3′ (forward) and 5′-TGTGGTCATGAGTCCTTCCACGAT-3′ (reverse) for human GAPDH.
5
RNA Extraction and RT-PCR Analysis
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Total cellular RNA was isolated using Trizol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in 3T3 cells following 48 h of transfection, according to the manufacturer's recommendations. All processes were conducted in a sterile and ribonuclease-free environment. Isolated RNA was dissolved in nuclease-free water and stored at −20°C. RT-PCR was performed using FastQuant cDNA first strand synthesis kit (Tiangen Biotech Co., Ltd., Beijing, China) using total RNA (1 µg) for 5 min at 42°C. The cDNA samples from RT reactions were amplified using 2X Taq PCR MasterMix (Tiangen Biotech Co., Ltd.) and the following primers: Sense, 5′-GGACGCGGGTCGAGTTTAAACAAGCCTG-3′, and anti-sense, 5′-CGAATTCTCACAGGGCAATGATCCC-3′. Thermal cycling conditions were as follows: Incubation at 50°C for 30 min and 94°C for 2 min, followed by 30 cycles of 94°C for 15 sec, 68°C for 30 sec and extension at 72°C for 10 min. The PCR products were loaded onto a 1% agarose gel containing SYBR® Safe DNA Gel Stain (Thermo Fisher Scientific, Inc.) for electrophoresis at 100 V for 40 min. The gel was then analyzed and photographed using the BIO-RAD Gel Doc XR + (Bio-Rad Laboratories, Inc.).
6
Verifying Alternatively Spliced DEGs by qRT-PCR
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The four groups randomly selected three alternatively spliced DEGs for Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) verification. Total RNA was extracted from ATII cells to synthesize cDNA using a FastQuant cDNA first-strand synthesis kit (TianGen, China). The cDNA was subjected to qRT-PCR analysis. Transcript-specific primers (Supplementary Table S1 in Supplementary material 1 ) were designed based on the unique regions of selected alternatively spliced DEGs using Primer 5.0 software, β-actin was used as reference genes, and expression levels were calculated using the 2−ΔΔCt method. PCR conditions were performed as follows: 95°C for 30 s, forty cycles at 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s.
7
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated from cells grown in fermentation medium using the Ultrapure Total RNA rapid extraction kit (Tiangen Biotech, Beijing, China). After extracting total RNA, the FastQuant cDNA first strand synthesis kit (Tiangen Biotech, Beijing, China) was used for reverse transcription, following the manufacturer’s instructions. qRT-PCR was carried out using SuperReal PreMix Plus (Tiangen Biotech, Beijing, China) with the following conditions: 95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s. Primers used are shown in Table S1 , and relative expression levels were calculated by the 2−ΔΔCt method using the 16 s rDNA as the internal control gene. All reactions were performed in triplicate.
8
Animal Tissue RNA Extraction and cDNA Synthesis
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Tissue RNA was extracted with the Animal Tissue Total RNA Extraction Kit (TIANGEN, DP431, CHINA). A 1μg total RNA was reversed transcribed into cDNA using the Fast Quant cDNA First-Strand Synthesis Kit (TIANGEN, KR106). RNA extraction and reverse transcription were performed according to the instructions.
9
Pulmonary Tissue Total RNA Extraction and qRT-PCR Analysis
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Total RNA from pulmonary tissues was extracted with a TRIzol reagent kit and reverse-transcribed into cDNA using a FastQuant cDNA first-strand synthesis kit (TianGen, China). SYBR® Premix Ex Taq™ II (TaKaRa, China) was used for real-time fluorescence quantitative analysis. In total, eight DEmRNAs and eight DEmiRNAs were randomly selected to determine sequencing accuracy. The primers used here were designed using Primer 5.0 software and are listed in Supplementary Tables 1 , 2 (Supplementary Material 1 ).
The experimental data were analyzed with the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) and SPSS 20.0 (SPSS, Chicago, IL, USA). The comparisons were conducted by one-way analysis of variance (ANOVA), and p < 0.05 was considered statistically significant.
The experimental data were analyzed with the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) and SPSS 20.0 (SPSS, Chicago, IL, USA). The comparisons were conducted by one-way analysis of variance (ANOVA), and p < 0.05 was considered statistically significant.
10
Cucumber Genomic DNA and RNA Isolation
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Genomic DNA was isolated from the two cucumber sister lines B21-a-2-1-2 and B21-a-2-2-2 seedling leaves with a Plant Genomic DNA kit (TianGen Biotech, China) according to the manufacturer’s instructions. Total RNA from various samples was isolated using RNAprep Pure Plant Kit, and cDNA was generated using a FastQuant cDNA first strand synthesis kit (TianGen Biotech, China) according to the manufacturer’s instructions. The DNA and RNA were detected on 1.2% agarose gels, and the purity of the DNA and RNA was determined by spectrophotometry (Biodrop).
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