The total RNA of the lung cancer cell lines was extracted with RNAprep Pure Cell Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. cDNA was then synthesized at 42°C for 15 minutes followed by heating to 95°C for three minutes to establish a library according to the instructions provided in the FastQuant cDNA First Chain Synthesis Kit (Tiangen Biotech).
Fastquant cdna first chain synthesis kit
Manufactured by Tiangen Biotech
Sourced in China
The FastQuant cDNA First Chain Synthesis Kit is a laboratory tool used for the reverse transcription of RNA to complementary DNA (cDNA). It provides the necessary reagents and protocols to efficiently synthesize the first strand of cDNA from total RNA or messenger RNA (mRNA) samples.
Automatically generated - may contain errors
Lab products found in correlation
Rnaprep pure cell kit, by Tiangen Biotech (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Mastercycler ep realplex4, by Eppendorf (1 mentions)
Superreal premix plus, by Tiangen Biotech (1 mentions)
Total rna extraction kit, by Tiangen Biotech (1 mentions)
Faststart universal sybr green master mix rox, by Roche (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Lightcycler 480ii, by Thermo Fisher Scientific (1 mentions)
Superreal premix color sybr green, by Tiangen Biotech (1 mentions)
6 protocols using fastquant cdna first chain synthesis kit
1
RNA Extraction and cDNA Synthesis
2
Quantitative RT-PCR for Liver Gene Expression
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Total RNA was isolated from liver tissues using TRIzol reagent (Tiangen Biotech, Beijing, China). One μg of total RNA was used to synthesize cDNA with the FastQuant cDNA First Chain Synthesis Kit (Tiangen Biotech, Beijing, China). The quantitative RT-PCR reaction volume was 20 μL, which included 1 μL template DNA, 10 μL SYBR® Premix Ex Taq™ (Takara, Otsu, Shiga, Japan), 7 μL ddH2O, and 1 μL of each forward and reverse primer. The reaction was initiated with the following conditions: 95 °C for 30 s (preincubation), 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control gene. The relative mRNA expression was calculated using the 2−ΔΔCT method. Table 2 shows the sequences of primers used in the current study.
3
Quantitative Gene Expression Analysis
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Total RNA extraction kit was used for total RNA extraction. Approximately 1,000 ng total RNA was reverse transcribed in 20 µl volume using FastQuant cDNA First Chain Synthesis kit (both from Tiangen) according to the manufacturer's instructions. Quantitative real-time PCR (qPCR) was performed using Super Real PreMix Plus (SYBR-Green) (Tiangen) on Eppendorf Mastercycler ep realplex4, with GAPDH as an internal control. The PCR program was as following: 3 min at 95°C, followed by 40 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 15 sec, and last step was the melting curve analysis program. All experiments were independently performed three times, and three replicates each time. The following primers were used: 5′-TCCTGCACCACCAACTGCTT-3′ (forward) and 5′-GGGGCCATCCACAGTCTTCT-3′ (reverse) for FIGNL1; 5′-CTCAGCGTGCATCAGGGTCT-3′ (forward) and 5′-CTGCTCTCCCCCATCTTGCT-3′ (reverse) for GAPDH; 5′-GTTCCTCCTTGGAAAGCAAACAGTA-3′ (forward) and 5′-CAGGGCATCTTCACGCTCTATTT-3′ (reverse) for cyclin A2; 5′-AGAAATGGCCAAAATCGA CA-3′ (forward) and 5′-CCCGGTCATCATCTTCTTTG-3′ (reverse) for cyclin E1; 5′-TATGCCTGATTACAAGCCAAGTTTC-3′ (forward) and 5′-GATAACAAGCTCCGTCCATCTTCAT-3′ (reverse) for CDK2; 5′-CATTGTTGTGTTTCACTGCGAGTTT-3′ (forward) and 5′-GGACATACAGCTCAGGGTAGTGGAG-3′ (reverse) for cdc25A. Relative gene expression was calculated by the 2−ΔCt method.
4
Total RNA Extraction and cDNA Synthesis
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Total RNA Extraction Kit (Tiangen Biotech) was used to extract the total RNA. Approximately 1,000 ng of total RNA was reverse transcribed in 20 µL volume using FastQuant cDNA first chain synthesis Kit (Tiangen Biotech) and the manufacturer’s instructions were followed throughout the operation. cDNA were stored at −20°C for future experiments.
5
Total RNA Extraction and qRT-PCR Analysis
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Add collected cells and tissues to TRIzol (Invitrogen, Carlsbad, CA, USA) to extract total RNA according to the RNAiso TM Plus (Takara, Japan) kit instruction, and the concentration of RNA is measured by a spectrophotometer (NanoDrop, Thermo Fisher Scientific, USA). Use FastQuant cDNA First Chain Synthesis Kit (Tiangen Biotech, Beijing, China) to conduct the reverse transcription, prepare the reaction system and expand (Applied Biosystems, Thermo Fisher Scientific, USA) according to the qRT-PCR kit Fast Start Universal SYBR Green Master Mix (Rox) (Roche Diagnostics GmbH Mannheim, Germany) instructions and analyze results.
6
qRT-PCR Analysis of R. solanacearum Virulence Genes
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R. solanacearum strains were grown to an OD600 of about 1.0 and total RNA were extracted by using a RNeasy Mini Kit (QIAGEN, Hilden, Germany). Contaminated genomic DNA was digested with DNaseI (Takara, Dalian, China) and confirmed by PCR using the primer pair for 16S rDNA. The cDNA was synthesized using the FastQuant cDNA first chain synthesis Kit (TIANGEN BIOTECH CO. LTD, Beijing, China) according to the manufacturer’s instructions. qRT-PCR was performed with Super Real PreMix Color SYBR Green (TIANGEN BIOTECH CO. LTD, Beijing, China) on Light Cycler480II (Applied Biosystems by Roche, Germany). The absolute value of –ΔΔCt = −(ΔCt1–ΔCt2) were calculated as described in the 2-ΔΔCt method (Livak and Schmittgen, 2001 (link)). Each assay was repeated from RNA isolation for three independent experiments with at least three replications per trial. All the Ct data of rmy genes, phcA and phcR in EP1 and related mutants have been listed in Supplementary Tables S2 -S4 .
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