Total RNA was isolated from patients' tissues and cell lines using TRIzol Total RNA Reagent (Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Complementary (c)DNA was synthesized from 2 µg total RNA using the RevertAid™ H Minus First Strand cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan) and cDNA was stored at −20°C. The RT reactions were terminated by inactivation at 95°C for 10 min and stored at 4°C. Primers used were obtained from Chang Jing Bio-Tech, Ltd. (Changsha, China) and were identical to those used in our previously published study (18 (link)); they are presented in Table II . qPCR was performed using the SYBR PrimeScript RT-PCR kit (Takara Bio, Inc.) in an Applied Biosystems 7500 Fluorescent Quantitative PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions used were as follows: 95°C for 30 sec; followed by 40 amplification cycles at 95°C for 5 sec and 60°C for 34 sec. The values were normalized to the internal control products of β-actin. Quantification of gene expression was performed using the 2−ΔΔCq method (26 (link)), with Cq representing the threshold cycle. Target gene expression per sample was determined using the following equation: Target in tumor tissue/target in non-tumorous tissue. All reactions were performed in triplicate and normalized to β-actin expression.
Revertaid h minus first strand cdna synthesis kit
Manufactured by Takara Bio
Sourced in Japan
The RevertAid™ H Minus First Strand cDNA Synthesis Kit is a reagent kit used for the synthesis of first-strand complementary DNA (cDNA) from RNA templates. The kit includes the necessary components for reverse transcription, including the RevertAid H Minus reverse transcriptase enzyme, reaction buffers, and other required reagents.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Sybr primescript rt pcr kit, by Takara Bio (4 mentions)
Applied biosystems 7500 fluorescent quantitative pcr system, by Thermo Fisher Scientific (4 mentions)
Trizol total rna reagent, by Thermo Fisher Scientific (2 mentions)
Cfx connect qpcr detection system, by Bio-Rad (2 mentions)
Sybr primescript qpcr kit, by Takara Bio (2 mentions)
Cell reagent rna protect, by Qiagen (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Itap universal sybr green supermix kit, by Bio-Rad (1 mentions)
One step sybr primescript rt pcr kit, by Takara Bio (1 mentions)
Sybr primerscript rt pcr kit, by Takara Bio (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Miscript reverse transcription kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
12 protocols using revertaid h minus first strand cdna synthesis kit
1
Quantitative Real-Time PCR for Gene Expression
2
Follicular Gene Expression Analysis
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To evaluate gene expression, follicles in the
experimental and control groups were collected in four
replicates: on day 0 (control group, 20 (link)-30 preantral
follicles in each replicate), and day 13 (experimental
groups, 15 (link)-20 antral follicles in each replicate) of
culture, pooled in Cell Reagent RNA Protect (RNeasy
kit, Qiagen, Germany) and stored at -70˚C until RNA
extraction. Total RNA was extracted from each of the
separated follicular pools using an RNeasy Micro Kit
(Qiagen, Germany) according to the manufacturer’s
instructions. cDNA was also synthesized using Revert
Aid H Minus First Strand cDNA Synthesis Kit (Takara,
Otsu, Shiga, Japan) and random hexamers according
to the manufacturer’s instructions.
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To evaluate gene expression, follicles in the
experimental and control groups were collected in four
replicates: on day 0 (control group, 20 (link)-30 preantral
follicles in each replicate), and day 13 (experimental
groups, 15 (link)-20 antral follicles in each replicate) of
culture, pooled in Cell Reagent RNA Protect (RNeasy
kit, Qiagen, Germany) and stored at -70˚C until RNA
extraction. Total RNA was extracted from each of the
separated follicular pools using an RNeasy Micro Kit
(Qiagen, Germany) according to the manufacturer’s
instructions. cDNA was also synthesized using Revert
Aid H Minus First Strand cDNA Synthesis Kit (Takara,
Otsu, Shiga, Japan) and random hexamers according
to the manufacturer’s instructions.
3
Quantifying ABCB4 Gene Expression
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Total RNA was extracted from the cells with TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The extracted RNA was reverse transcribed into cDNA using the Revert Aid™ H Minus First Strand cDNA Synthesis kit (Takara Bio, Inc.) according to the manufacturer's protocols. qPCR was performed using the SYBR® Green PCR Master mix (Takara Bio, Inc.). The following primer pairs were used for the qPCR: ABCB4, forward 5′-TGGCCCTGGTTGGAAGTAGTG-3′ and reverse 5′-AGAAGGATCTTGGGGTTGCGAA-3′; and β-actin, forward 5′-GGATGCAGGAGATCACTG-3′ and reverse 5′-CGATCCACACGGAGTACTT-3′. qPCR was conducted as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Relative quantification of ABCB4 mRNA was analyzed using the 2−ΔΔCq method relative to the β-actin expression level in each sample (33 (link)).
4
Gene Expression Analysis via qPCR
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Total RNA was extracted using the Trizol Total RNA Reagent (Invitrogen) following the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized with 2 μg total RNAs using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Takara, Otsu, Japan). All primers were obtained from GenePharma (Shanghai, People’s Republic of China), and the sequences we used were listed in Table 1 . Real-time quantitative polymerase chain reaction (PCR) was analyzed using the SYBR PrimeScript RT-PCR kit (Takara) in an Applied Biosystems 7500 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The cycling conditions were as follows: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, and 60°C for 34 s.
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA were extracted using the TRIzol reagent (Thermo Fisher Scientific), and reverse transcribed into complementary DNA (cDNA) using the RevertAid H Minus First Strand cDNA synthesis kit (Takara, Tokyo, Japan). Quantitative reverse transcription PCR was performed using the iTap Universal SYBR Green Supermix kit (Bio‐Rad, Hercules, CA, USA). The amplification conditions were: initial denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s, and extension at 72 °C for 30 s. Relative expressions of target genes were determined using the comparative Ct (2−ΔΔCt) method with Gapdh as the internal standard. The primer sequences used in this assay are shown in Table S2 , Supporting Information.
6
Profiling of lncRNA and miRNA Expression
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Total RNA of cultured cells was prepared with the TRIzol reagent (Invitrogen, CA, USA). The AMV Reverse tTanscriptase XL (Takara Bio, Otsu, Japan) and RevertAid™ H Minus First Strand cDNA Synthesis Kit (Takara) were used for reverse transcription. The expression levels of lncRNA and micRNA were measured with the One Step SYBR Prime Script RT-PCR Kit (Takara Bio, CA, USA). The GAPDH and U6 were used as their endogenous controls. All experiments were independently repeated at least three times. The sequences of the primers are listed in the Supplementary Table 1 .
7
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from the tissues or cells using the Trizol reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. The RNA samples were reverse-transcribed into cDNA with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Takara, Otsu, Japan). β-Actin was used as the internal control. All primers used for qPCR were obtained from GenePharma (Shanghai, China), and the sequences are listed in Table 2 . qPCR was conducted with the SYBR PrimeScript qPCR kit (Takara) in a CFX Connect™ qPCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. All samples were analyzed in triplicate. Fold changes of ALK or lincROR were calculated by the relative quantification (2−ΔΔCt) method.
8
Osteosarcoma Tumor RNA Expression Analysis
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Total RNA was isolated from the osteosarcoma tumor tissues, matched adjacent normal tissues and osteosarcoma cells using TRIzol® reagent (Invitrogen Life Technologies). cDNA synthesis was performed with 2 μg total RNA using the RevertAid™ H Minus First Strand cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan). The primers were obtained from Sheng Gong (Shanghai, China), and the sequences are included in Table II . A qPCR was performed using the SYBR® PrimeScript RT-PCR kit (Takara Bio, Inc.) and the Applied Biosystems 7500 Fluorescent Quantitative PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA). The reaction mixtures were incubated at 95°C for 30 sec, followed by 40 amplification cycles of 95°C for 5 sec and 60°C for 34 sec. The comparative cycle threshold method was used to quantify the relative expression levels of mRNA and lncRNA (19 (link)). Expression levels of the housekeeping gene β-actin were used to normalize the expression levels of the genes-of-interest. The expression levels of the genes were calculated in each patient using the following ratio: target in cancerous tissue/target in non-cancerous tissue [R(C/N)].
9
Quantitative Real-Time PCR for CRC
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Total RNA was extracted from CRC tumor tissues and matched adjacent normal tissues, using Trizol Total RNA Reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. The first strand complementary DNAs (cDNAs) were synthesized with 2 μg total RNAs by means of RevertAidTM H Minus First Strand cDNA Synthesis Kit (Takara, Otsu, Japan). The primer sequences were designed and synthesized by GenePharma (Shanghai, People’s Republic of China). Quantitative real-time PCR was performed using the SYBR PrimeScript RT-PCR kit (Takara) using Applied Biosystems 7500 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s recommendations. The reaction conditions were designed as follows: 1 cycle at 95°C for 30 seconds followed by 40 cycles at 95°C for 5 seconds, and 60°C for 34 seconds. The ΔΔCT calculation with CT served as the threshold cycle for quantification of gene expression, and the target gene expression level in patients was measured as “ratio (target in tumor tissue/target in nontumorous tissue”: R (T/N)). All reactions were performed in triplicate and normalized by the internal control products of GAPDH. All sequences we used are listed in Table 2 .
10
Se-enriched Cordyceps militaris Modulates Cell Cycle and Apoptosis
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Total RNA was extracted from NCI-H292 and A549 cells treated with varying concentrations of Se-enriched Cordyceps militaris (0, 12.5, 25, and 50 mg/mL for A549 cells, and 0, 4, 8, and 12 mg/mL for NCI-H292 cells) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s instruction. 1 μL of total RNA was used to synthesize complementary DNA (cDNA) using the RevertAidTM H Minus First Strand cDNA synthesis Kit (Takara, Otsu, Japan). qPCR analysis was conducted using SYBR PrimerScript RT-PCR kit (Takara, Biotechnology, Inc., Dalian, People’s Republic of China) on a CFX Connect™ Real-Time PCR Detection System (BIO-RAD Laboratories, Inc., California, USA). GAPDH was used as the internal control. Fold changes of genes were calculated by the relative quantification (2−ΔΔCt) method. The sequences of the primers were listed as follows: cyclin B1, 5ʹ-CCCCTGCAGAAGAAGACCTG-3ʹ (forward) and 5ʹ-TGACTGCTTGCTCTTCCTCA-3ʹ (reverse); CDK1, 5ʹ-CCCTTTAGCGCGGATCTACC-3ʹ (forward) and 5ʹ-CATGGCTACCACTTGACCTGT-3ʹ (reverse); BCL-2, 5ʹ-CCGCGACTCCTGA TTCATTG-3ʹ (forward) and 5ʹ-AGTCTACTTCCTCTGTGATGTTGTA-3ʹ (reverse); BAX, 5ʹ-AAACTGGTGCTCAAGGCCC-3ʹ (forward) and 5ʹ-AAAGTAGGAGAGGAGGCCGT-3ʹ (reverse); GAPDH, 5ʹ-ACGGATTTGGTCGTATTGGGCG-3ʹ (forward) and 5ʹ-GCTCCTGGAAGATGGTGATGGG-3ʹ (reverse).
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