To validate the up-regulated expression of HSP70 and lysozyme in 3-day regenerated tissue, 300 tail-amputated E. fetida were equally divided to 6 groups for different regeneration days (0-day, 12-h, 1-day, 2-day, 3-day and 4-day). RNA isolation was performed in each group with the same instructions in RNA-Seq analysis. cDNA was synthesised using BeyoRT™ II cDNA first strand synthesis kit (Beyotime Institute of Biotechnology, China) according to manufacturer's instructions. A final 20 μL cDNA was generated from 4 μg of RNA per sample. PCR amplification was performed with 0.1–1 μg final concentration of cDNA template, 0.8 μM Primer Mix (Table 6 ) and 1⨯ PCR Master Mix (Beyotime, China) on Mastercycler Pro PCR (Eppendorf AG., Germany) using the following cycling condition: 94 °C for 3 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed by 72 °C for 10 min. The PCR products were analyzed on 1% agarose gel (Beyotime, China) with Sub-Cell GT cell (Bio-Rad Laboratories, Inc., U.S.A) under 100 V for 40 min. The results were analyzed by ImageJ version k 1.45 with the reference gene β-actin and the ratio of target genes/β-actin was calculated. A paired Student's t-test by using Prism 6 software was performed to determine the significance between different groups (P < 0.05) from three individual experiments.
Beyort 2 first strand cdna synthesis kit
Manufactured by Beyotime
Sourced in China
The BeyoRT™ II First Strand cDNA Synthesis Kit is a laboratory tool used for the reverse transcription of RNA into complementary DNA (cDNA). The kit provides the necessary reagents and components to efficiently synthesize the first strand of cDNA from total RNA or mRNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Beyotime (4 mentions)
Trizol, by Beyotime (2 mentions)
Beyofast sybr green qpcr mix, by Beyotime (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Pcr master mix, by Beyotime (1 mentions)
Sub cell gt cell, by Bio-Rad (1 mentions)
Pcr mastercycler pro, by Eppendorf (1 mentions)
Pcr instrument, by Bio-Rad (1 mentions)
Mirna first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
Trizol reagent, by Solarbio (1 mentions)
Trizol, by Merck Group (1 mentions)
Axyprep total rna extraction kit, by Corning (1 mentions)
Applied biosystems tm quant studiotm3 5, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq, by Takara Bio (1 mentions)
Rnaeasy animal rna isolation kit with spin column, by Beyotime (1 mentions)
Sybr green qpcr mix, by Beyotime (1 mentions)
Sybr green reagent, by Takara Bio (1 mentions)
22 protocols using beyort 2 first strand cdna synthesis kit
1
Validating HSP70 and Lysozyme in Regenerated Tissue
2
Quantitative Real-Time PCR Analysis of FDX1 and SLC27A5
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HepG2 and LM-3 cells were seeded into 6-well plates and conduct normal culture. Cells are collected when their density reaches 80%. TRIzol (AN51L758, Life-iLab) was used to lyse cells to obtain total RNA. BeyoRT™ II cDNA First Strand Synthesis Kit (D7168M, Beyotime) was used to obtained cDNA. 2x qPCR Mix (AN19L918, Life-iLab) was used to perform qPCR. The primers of target genes are as follows:
FDX1 forward, 5’-GTTCAACCTGTCACCTCATCTT-3’;
FDX1 Reverse 5’-CCAACCGTGATCTGTCTGTTAG-3’;
SLC27A5 forward, 5’-AGAGGACCGGACACATACA-3’;
SLC27A5 Reverse 5’-GTAGACTTCCCAGATCCGAATAG-3’;
GAPDH forward, 5’-GTCAAGGCTGAGAACGGGAA-3’;
GAPDH Reverse 5’-AAATGAGCCCCAGCCTTCTC-3’.
FDX1 forward, 5’-GTTCAACCTGTCACCTCATCTT-3’;
FDX1 Reverse 5’-CCAACCGTGATCTGTCTGTTAG-3’;
SLC27A5 forward, 5’-AGAGGACCGGACACATACA-3’;
SLC27A5 Reverse 5’-GTAGACTTCCCAGATCCGAATAG-3’;
GAPDH forward, 5’-GTCAAGGCTGAGAACGGGAA-3’;
GAPDH Reverse 5’-AAATGAGCCCCAGCCTTCTC-3’.
3
Quantitative RT-PCR Analysis of Gene Expression
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Total RNAs from colon tissues and RAW264.7 cells were isolated using the TRIzol reagent (R1100, Solarbio). Subsequently, cDNAs were synthesized using the BeyoRT™ II cDNA first‐strand synthesis kit (D7168S, Beyotime) or miRNA cDNA first‐strand synthesis kit (KR211, TIANGEN). Then, we used the 2×Taqman PCR MasterMix (SR2110, Solarbio) and PCR instrument (CFX96, Bio‐Rad) to generate qRT‐PCR reactions. The results were normalized to GAPDH or U6, and the 2−ΔΔCt method was utilized to count the mRNA expression levels.27 Primers are shown in Table 1 .
4
Extraction and Quantification of RNA
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Total RNA was isolated using TRIzol (Sigma-Aldrich, St Louis, MO, USA) at 48 h post-transfection. Then, 5 μl of RNA sample was diluted 20 times with RNase-free ultrapure water. The concentration and purity of RNA were determined by reading an optical density (OD) value at 260 and 280 nm. As per the instructions of BeyoRT™ II cDNA First-Strand Synthesis Kit (Beyotime, Shanghai, China), reverse transcription was This website utilizes technologies such as cookies to enable essential site functionality, as well as for analytics, personalization, and targeted advertising. To learn more, view the following link: Privacy Policy performed in a PCR amplification instrument to synthesize complementary DNA (cDNA). The primers generated by Sangon (Shanghai, China) are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for CDC20, and U6 was used as an internal control for miR-1321 and miR-7515.
5
Quantifying Gene Expression in Bone Marrow Cells
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Total RNA was extracted from BM specimens using the PAXgene Bone Marrow RNA Kit (QIAGEN) following the manual. Tumors were homogenized in liquid nitrogen and resuspended in TRIzol reagent (Beyotime) for RNA extraction. BMSCs, BMMNCs and AML cells were washed and resuspended in TRIzol reagent, and total RNA was isolated and subjected to RNA quantification. Subsequently, RNA was reversely transcribed into cDNA using the BeyoRT™ II First Strand cDNA Synthesis Kit (Beyotime). Quantitative PCR was applied to detect the expression of Bmal1, EBF3, EZH2 and ALOX15. Gene expression was normalized to β‐actin, and the 2−ΔΔCt method was used for calculation. Primers are shown in Table S2 .
6
Quantifying Gene Expression in PANC-1 Cells
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Total RNA was extracted from PANC-1 cells treated without or with 200 or 400 μM ISO-1 for 24 hours using AxyPrep Total RNA Extraction Kit (Axygen Inc, Corning Inc) in accordance with manufacturer’s instructions. Complementary DNA (cDNA) was then synthesized using 2 μg of purified RNA using the BeyoRT II First Strand cDNA Synthesis Kit (Beyotime Biotechnology) and subsequently used as template for qPCR in reaction mixture containing specific forward and reverse primers, and TB Green Premix Ex Taq (Takara Bio Inc., Shiga, Japan). qPCR was performed on a Applied Biosystems TM Quant StudioTM3&5 (Thermo Fisher Scientific) with the following cycling conditions: 95 °C for 30 secs; followed by 40 cycles of 95 °C for 5 secs, 60 °C for 30 secs. Specific primers against the following human genes were used: MIF (Sense: 5′-GGACAGGGTCTACATCAACTA-3′, and Anti-Sense: 5′-TCTTAGGCGAAG GTGGAG-3′); TNF-α (Sense: 5′-TTATTTATTTACAGATGAATG-3′, and Anti-Sense: 5′-TTAGACAACTTAATCAGA-3′); NF-κB p65 (Sense: 5′-CCTTATCAAGTGTCTTCCATCA-3′, and Anti-Sense: 5′-AATGCCAGTGCCATACAG-3′); and GAPDH (Sense: 5′-CTCTGGTA AAGTGATATTGT-3′, and Anti-Sense: 5′-GGTGGAATCATATTGGAACA-3′). The expression of target genes was normalized to internal housekeeping gene GAPDH using the 2−ΔΔCT method.
7
RNA Extraction and RT-qPCR Analysis from Lung Tissue
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For total RNA extraction, Trizol (R0016, Beyotime) was used for the RNA extraction from mice lung tissue, and RNAeasy™ Animal RNA Isolation Kit with Spin Column (R0027, Beyotime) was used for the RNA extraction from primary mouse lung fibroblasts and HFL1 cells. A Nanodrop machine was used to determine the concentration and purity of extracted RNA. BeyoRT™II First Strand cDNA Synthesis Kit (D7168L, Beyotime) was used to reverse messenger RNA (mRNA) to complementary DNA (cDNA). Then, real-time quantitative polymerase chain reaction (RT-qPCR) was performed using BeyoFast™ SYBR Green qPCR Mix (D7260, Beyotime). All experiments in this part were carried out following the manufacturer’s instructions, and all data were normalized to the expression level of β-actin mRNA. The primers’ sequence used in RT-qPCR is listed in Table 1 .
8
Liver RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from liver tissues and LX-2 cells using a total RNA kit (Beibei Biotechnology, Zhengzhou, China). A BeyoRT™II First Strand cDNA Synthesis Kit (Beyotime, Shanghai, China) was used to reverse transcribe the RNA (1–5 μg). SYBR Green qPCR mix (Beyotime, Shanghai, China) and a QuantStudio5 system (ABI, Thermo Fisher Scientific, Waltham, MA, USA) were utilized for qRT-PCR. The cDNA was amplified using the given procedure: 50 °C for 2 min, 95 °C for 2 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. The mRNA expression level of the desired genes was standardized against GAPDH using the 2−ΔΔCt method. Primer sequences are listed in Supplementary Table S2 .
9
Quantification of Liver mRNA Expression
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Total mRNA was isolated from the liver using TRIzol Reagent (Qiagen China Co., Ltd., Shanghai, China). RT was performed using the BeyoRT™ II First Strand cDNA Synthesis Kit (Beyotime Institute of Biotechnology) and then qPCR was performed using the Light-Cycler 480 System (Roche Diagnostics, Basel, Switzerland) with SYBR Green reagent (Takara Biotechnology, Co., Ltd., Dalian, China). The amplification reactions were according to the following thermal cycling conditions: 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The quantification was expressed as the ratio of target genes to GAPDH mRNA using the 2−ΔΔCq method (16 (link)). RT-qPCR was performed using the primers in Table I .
10
Bavachin Modulates Virulence Genes
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The effect of bavachin on transcript levels of hla, RNAIII, and agrA was examined by RT-qPCR. After treatment with different concentrations of bavachin or DMSO overnight at 37°C, with shaking until the OD600 reached 2.5, total RNA was extracted by the TRIzol method. The BeyoRT II First Strand cDNA Synthesis Kit (Beyotime) was used for cDNA synthesis. qPCR was performed using NovoStart SYBR qPCR SuperMix Plus (Novoprotein, China). 16sRNA was used as the reference gene and the primer sequences used were as follows: 16S forward primer 5’-TGATCCTGGCTCAGGATGA-3’, 16S reverse primer 5’-TTCGCTCGACTTGCATGTA-3’; hla forward primer 5’-GGTATATGGCAATCAACTT-3’, hla reverse primer 5’-CTCGTTCGTATATTACATCTAT-3’; RNAIII forward primer 5’-AATTAGCAAGTGAGTAACATTTGCTAGT-3’, RNAIII reverse primer 5’-GATGTTGTTTACGATAGCTTACATGC-3’; agrA forward primer 5’-GCAGTAATTCAGTGTATGTTCA-3’, agrA reverse primer 5’-TATGGCGATTGACGACAA-3’. The expression of genes was obtained by calculating 2-ΔΔCt after normalizing the cycle threshold (Ct) of each gene with the Ct value of the housekeeping gene (16S RNA).
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