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Primerscript reagent kit

Manufactured by Takara Bio
Sourced in China, Japan

The PrimerScript™ reagent kit is a high-performance reverse transcription (RT) kit designed for efficient and reliable cDNA synthesis from RNA samples. The kit includes a thermostable reverse transcriptase enzyme, RNase inhibitor, and optimized reaction buffers.

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14 protocols using primerscript reagent kit

1

Liver RNA Extraction and qPCR Analysis

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Trizol Reagent (Vazyme, NanJing, China) was used to extract total RNA from liver tissue, which was then treated by deoxyribonuclease I to remove the contaminant DNA. RNA was quantified based on the absorption of light by a Nanodrop ND-2000c spectrophotometer (Thermo Scientific, Camden, UK) at 260 nm (A260) and 280 nm. From each sample, 1 μg of RNA was used to synthesize cDNA in a 20 μL reaction mixture using the Primer-ScriptTM reagent kit (TaKaRa, Dalian, China) according to the manufacturers’ instructions. The real-time quantitative polymerase chain reaction was carried out by using the SYBR Premix Ex Taq II kit (TaKaRa) in an ABI 7300 fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA). The 20 μL reaction system included 10 μL of SYBR Premix Ex Taq buffer, 0.4 μL each of forward and reverse primers and dye, 2 μL of cDNA template, and 6.8 μL of distilled water. The real-time PCR cycling conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The relative mRNA expression was determined using β-actin as an internal reference gene. The significance and correlation of quantitative results were analyzed by using 2‒ΔΔct as per Livak and Schmittgen [19 (link)]. Primer sequences are shown in Table 2.
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2

Liver RNA Extraction and RT-qPCR Analysis

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Total RNA was extracted from liver samples using the Trizol reagent (TaKaRa Biotechnology Co., Dalian, China), according to the manufacturer’s instructions. The concentration of RNA in the final preparations was measured with a Nanodrop ND-2000c spectrophotometer (Thermo Fisher Scientific, Camden, USA). Reverse transcription was immediately performed using the Primer-ScriptTM reagent Kit (Takara Biotechnology Co., Dalian, China).
The expression of genes in liver was measured using real-time quantitative PCR (RT-qPCR) with SYBR Premix Ex Taq II kit (Tli RNaseH Plus; Takara Biotechnology) and an ABI 7300 Fast Real-Time PCR detection system (Applied Biosystems). The 20 μL reaction system included 2 μL of cDNA template, 0.4 μL of ROX reference dye (50×), 10 μL of SYBR Premix Ex Taq (2×), 0.4 μL of each forward and reverse primer, and 6.8 μL of double-distilled H2O. The RT-qPCR cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. All of the samples were run in triplicate, and the mRNA expression level of genes were calculated using the 2−ΔΔCt method and normalized to the value of the reference gene β-actin. The final result of each target gene expression was expressed as the percentage of the CONT group. Primer sequences are shown in Table 2.
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3

Quantification of Tight Junction Proteins

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Samples of ileal mucosa were scraped on ice, frozen in liquid nitrogen immediately and stored at -80°C. Total RNA was extracted with Trizol reagents according to manufacturer’s instruction (Invitrogen, USA). RNA was quantified by nano-drop 2000 (absorbance ratios of 260/280 nm and 260/230 nm between 1.90 and 2.05) and verified by agarose gel electrophoresis. Then cDNA was prepared with Primer-ScriptTM reagent kit, provided by TakaRa Biotechnology Co. Ltd (Dalian, China).
The gene-specific primers of occludin, claudin-2 and ZO-1 were synthesized by Invitrogen Biotech Co. Ltd (Shanghai, China) and are listed in Table 2. GAPDH was chosen as a housekeeping gene. Reverse transcription polymerase chain reaction (RT-PCR) tests were conducted with ABI 7600 RT-PCR system with a SYBR Premix Ex TaqTM kits (TakaRa Biotechnology Co. Ltd Dalian, China). The relative mRNA expression was analyzed with ABI software and calculated with 2-ΔΔCt as described previously [39 (link)].
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4

Quantification of miR-665 and CRIM1 mRNA

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RNAios Plus (Takara Bio Inc., Shiga, Japan) was used to extract total RNA from cell lines and tissues, according to the manufacturer’s instructions. Reverse transcription and qRT-PCR of miR-665 were performed using the Hairpin-it™ miRNA RT-PCR Quantitation Kit (Gene Pharma, Shanghai, China), with U6 RNA as the internal reference. RNA reverse transcription was synthesized with the PrimerScriptTM reagent kit (Takara, Dalian, China) and SYBR Green (Solarbio, Beijing, China) was utilized to analyze the CRIM1 mRNA expression level, where glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an endogenous control. The Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, US) was used to perform qRT-PCR. All primers were as follows: miR-665 sense, 5′-GGTGAACCAGGAGGCTGAGG-3′, miR-665 antisense, 5′-CAGTGCAGGGTCCGAGGTAT-3′, U6 sense, 5′-CGCTTCGGCAGCACATATAC-3′, U6 antisense, 5′-TTCACGAATTTGCGTGTCATC-3′, CRIM1 sense, 5′- AGTTTCCAAGTCAGGATATGTGC-3′, CRIM1 antisense, 5′- AGCATAACCCTCGATCAGAACA-3′, GAPDH sense, 5′-AGCCACATCGCTCAGACTC-3′, GAPDH antisense, 5′- GCCCAATACGACCAAATTC −3′.
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5

Quantitative PCR Analysis of HMGB1 Expression

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The total RNA was extracted using TRIzol Reagent (Invitrogen) and the cDNA was obtained using the PrimerScript™ Reagent Kit (Takara, Dalian, China). Fluorescence quantitative PCR detection was carried out according to the instructions. The reaction system was 10 μl, and the reaction procedure was 94°C 3 min, 94°C 30 s, 60°C 20 s, 72°C 1 min, 40 cycles. The sequence was as follows: HMGB1F: 5′- -ACATAAATTCAAGAAAGGTGAT-3′, R: 5′-ATATGCTAAAATGTCTGCTTC-3′. The primer sequences of β-actin are F: 5′- CGCTCTCTGCTCCTCCTGTTC-3′, R: 5′- -ATCCGTTGACTCCGACCTTCAC-3′; the primer sequences of 1224-5p are F: 5′-GGAGCAGCATTGTACAGG-3′, R: 5′-CAGTGCGTGTCGTGGA-3′. The U6 primer sequences are F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′. Levels of gene expression were calculated by the 2-△△Ct method.
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6

Comprehensive RNA Extraction and qRT-PCR Protocol

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In this study, total RNA extraction of maintained cells or frozen tissues was
implemented by TRIZOL reagent (Invitrogen). After that, the PrimerScript™
reagent kit (TaKaRa, Shiga, Japan) was usedto obtain cDNA. Then, we implement
SYBR Premix Ex Taq II (TaKaRa) to exert the qRT-PCR following the manufacturer’s
instructions. GAPDH and U6 were used as the internal reference. All Primer
sequences were designed by RiboBio (Guangzhou, China). Primer sequences of
related genes for RT-qPCR:

ATF6: forward 5’-CCAGCAGAAAACCCGCATTC-3’ and reverse

5’-AACTTCCAGGCGAAGCGTAA-3’;

GRP78: forward 5’-AACCCAGATGAGGCTGTAGCA-3’ and reverse

5’-ACATCAAGCAGAACCAGGTCAC-3’;

β-catenin: forward 5’- TGACAAAACTGCTAAATGACGAGG -3’ and reverse

5’-CGCATGATAGCGTGTCTGGA-3’;

c-myc: forward 5’- CCACGAAACTTTGCCCATAG -3’ and reverse

5’- TGCAAGGAGAGCCTTTCAGAG-3’;

Cyclin D1: forward 5’- TGTCCCACTCCTACGATACGC -3’ and reverse

5’- CAGCATCTCATA AACAGGTCACTA C-3’;

LAMC1: forward 5′-ATTTCAATCAACCGCTCT-3’ and reverse

5’-GTTATGGACCTCCTTCGT -3’;

GAPDH: forward 5’-GACGTAGGGAGTGAAGGTC-3’ and reverse

5’-GAGAGTTCAGATGTTGATGG-3.’

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7

Quantitative Analysis of miRNA Expression

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Total RNA was isolated by using TRIzol Reagent (Invitrogen). The cytoplasmic and nuclear RNAs of the cells were separated and extracted by using a Paris Kit (Invitrogen). First-strand cDNA was then generated by using a PrimerScript™ reagent kit (TaKaRa, Dalian, China). PCR amplification was carried out by using a SYBR Green PCR kit (TaKaRa) on an ABI PRISM 7300 Sequence Detection system (Applied Biosystems, Foster City, CA, USA). The PCR primer sequences were as follows: miR-224 primer: 5′-GAGCCCAAGTCACTAGTGGT-3′, downstream primer: 5′-GTGCAGGGTCCGAGGT-3′; U6: sense strand 5′-GGCTGGTAAGGATGAAGG-3′, antisense strand 5′-TGGAAGGAGGTCATACGG-3′; GADPH upstream primer: 5′-AGCCACATCGCTCAGACA-3′, downstream primer: 5′-TGGACTCCACGACGTACT-3′. Gene expression in each group was calculated in accordance with the 2−ΔΔCT method. GAPDH was used as an internal reference to calculate relative total RNA expression, and U6 was used as an internal reference to calculate relative miRNA expression.
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8

Quantitative Expression Analysis of Genes and miRNAs

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Total RNA of cells and tissues was extracted using TRIzol reagent and reverse transcribed into cDNA using the PrimerScript™ reagent Kit (TaKaRa, Dalian, China). qRT-PCR experiments were conducted with the SYBR Premix Ex Taq (TaKaRa, Dalian, China) on the ABI StepOne™ Real-Time PCR system (Applied Biosystems, CA). The quantification of miRNAs was detected using miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany). Relative expression values of genes were analyzed by the 2−ΔΔCt method using β-actin and U6 for standardization. The primer sequences used for qRT-PCR are listed in Table S1.
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9

Skin Tissue RNA Extraction and qPCR Analysis

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Total RNA was isolated from skin tissue using TRIzol reagent (Takara, China) and reverse-transcribed into cDNA using a Primer Script™ Reagent Kit (Takara, China) QPCR. QPCR was carried out on an ABI 7300 system (ABI, USA) using FastStart Universal SYBR Green Master according to the manufacturer’s instructions. The thermal cycling conditions used for QPCR were 95°C for 5 min, followed by 45 cycles of 95°C for 30 s and 60°C for 45 s. Experiments were carried out using three biological and technical replicates, respectively. Data were expressed as mean±standard error.
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10

Quantitative Gene Expression Analysis

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Total RNA was isolated using TRIzol Reagent (Invitrogen). Cytoplasm and nuclear RNA of cells were separated and extracted using the PARIS Kit (Invitrogen). First strand cDNA was then generated using the PrimerScript™ reagent Kit (TaKaRa, Dalian, China). PCR amplification was carried out using a SYBR Green PCR kit (TaKaRa) on an ABI PRISM 7300 Sequence Detection system (Applied Biosystems, Foster City, CA, USA). Relative quantification of gene expression was calculated by the 2−ΔΔCt method,10 (link) with GAPDH or U6 as an endogenous control. All experiments were repeated in independent triplicate.
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