Forward and reverse primers

Manufactured by Takara Bio

Sourced in Japan, China

Forward and reverse primers are short, single-stranded DNA sequences used in the polymerase chain reaction (PCR) to amplify specific regions of DNA. They are designed to bind to complementary sequences on the DNA template, allowing the DNA polymerase enzyme to replicate the target sequence.

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Lab products found in correlation

Trizol, by Thermo Fisher Scientific (2 mentions) Accuprep genomic dna extraction kit, by Bioneer (1 mentions) Dntps, by Takara Bio (1 mentions) Taq polymerase, by Takara Bio (1 mentions) Accuprep pcr purification kit, by Bioneer (1 mentions) Thermo scientific nanodrop one microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Iq sybr green supermix, by Bio-Rad (1 mentions) Cfx connect real time pcr detection system, by Bio-Rad (1 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions) Superscript 3 kit, by Thermo Fisher Scientific (1 mentions) Quantstudio 5, by Thermo Fisher Scientific (1 mentions) Sybr premix ex taq tli rnaseh plus, by Takara Bio (1 mentions) Primescript rt master mix, by Takara Bio (1 mentions) Quantstudio 6 flex real time pcr detection system, by Thermo Fisher Scientific (1 mentions) Reverse transcriptase kit, by Takara Bio (1 mentions) Rnaiso plus, by Takara Bio (1 mentions) Sybr green mix, by Takara Bio (1 mentions) Tb green premix ex taq 2, by Takara Bio (1 mentions) Thermal cycler dice real time system 3, by Takara Bio (1 mentions)

Spelling variants of this product

Reverse primer (6 citations) Forward primer (5 citations) Forward and reverse primers for targeted mRNA (2 citations)

9 protocols using forward and reverse primers

Mitochondrial COI Gene Amplification from Spiny Lobster

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Genomic DNA was extracted from the muscle of the pereiopod of all 17 spiny lobsters. DNA was extracted using the AccuPrep^® Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea), following the manufacturer’s instructions. Concentration and purity of the extracted DNA were measured using a Thermo Scientific™ NanoDrop™ One microvolume UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
A polymerase chain reaction (PCR) was performed to amplify the mitochondrial COI gene region using the HCO1490/LCO2198 universal primers, specially designed for invertebrates (Folmer et al., 1994 (link)). PCR was performed using a 50 µL reaction mixture, consisting of 100 ng genomic DNA, 0.25 µL Taq polymerase (Takara Bio Inc., Shiga, Japan), 5 µL 10X Ex. Taq DNA polymerase buffer (Takara Bio Inc.), 1 µL each of 10 µM forward and reverse primers, and 4 µL (2.5 mM) dNTPs (Takara Bio Inc.). The PCR thermal profile comprised an initial step of 5 min at 94 °C, followed by 30 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s, followed by a final extension at 72 °C for 5 min. The amplified PCR products were separated using 1% agarose gel electrophoresis, and target bands were purified using the AccuPrep^® PCR Purification Kit (Bioneer), according to the manufacturer’s instructions.

Hettiarachchi S.A., Hyeon J.Y., Mahardini A., Kim H.S., Byun J.H., Kim H.J., Jeong J.G., Yeo J.K., Kim S.K., Kim S.J., Heo Y.S., Sathyadith J., Kang D.H, & Hur S.P. (2022). DNA barcoding and morphological identification of spiny lobsters in South Korean waters: a new record of Panulirus longipes and Panulirus homarus homarus. PeerJ, 10, e12744.

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Quantifying Aquaporin and PLD2 Expression

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Total RNA was extracted from A431 cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) at 24 h post-siRNA transfection. cDNA was synthesized from the isolated RNA using a RevertAid First Strand cDNA synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.
The qPCR assay was performed on a CFX Connect Real-Time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol. The qPCR analysis was performed with 25 µl (final volume) reaction mixture, containing 10.5 µl of cDNA, 12.5 µl of IQ SYBR Green Supermix (Bio-Rad Laboratories, Inc.) and 1 µl each of 10 µM forward and reverse primers (Takara Bio, Inc., Kusatsu, Otsu, Japan). The following primers were used for amplification of AQP3, PLD2 and the internal control (GAPDH): AQP3, forward 5′-CCCCTCTGGACACTTGGAT-3′ and reverse 5′-CACGAAGACACCCGCAAT-3′. PLD2, forward 5′-GCCTTGGGCATCAACAGT-3′ and reverse 5′-AGGTCAGTCAGTCGGTAGTG-3′. GAP DH, forward 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse 5′-AGGGGCCATCCACAGTCTTC-3′. Thermal cycling was initiated with an initial denaturation step at 50°C for 3 min, 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec, 61°C for 20 sec and 72°C for 20 sec. The 2^−ΔΔCq method (16 (link)) was used for data analysis, with results representative of three independent experiments.

Wang X., Tao C., Yuan C., Ren J., Yang M, & Ying H. (2017). AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma. Molecular Medicine Reports, 16(2), 1964-1972.

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Stem-loop qRT-PCR for miRNA Expression

Cited 1 time

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We used stem-loop qRT-PCR to confirm the miRNA expression levels^{71 (link)}. For selected miRNAs, ~1 μg DNA-free total RNA was hybridised with miRNA-specific stem-loop RT primers. The hybridised molecules were reverse transcribed into cDNAs using a Superscript III kit (Thermo Fisher Scientific). We designed forward miRNA-specific primers for the mature miRNA sequences and used a universal reverse primer for the stem-loop sequences. Reactions were repeated three times for each sample set. Each 20-μL reaction mixture contained 1 μL cDNA, 10 μL 2 × FastStart SYBR Green (Roche) and 0.8 μL forward and reverse primers (TaKaRa, Ohtsu, Japan). The PCR amplification conditions were as follows: 95 °C for 10 s and 60 °C for 30 s. The PCRs were conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Data were analysed using the 2^−ΔΔCt method to calculate relative gene expression^{72 (link)}. Supplementary Table S8 lists all primers used in the qRT-PCR experiments, including those for miRNAs and their targets.

Luo X., Cao D., Zhang J., Chen L., Xia X., Li H., Zhao D., Zhang F., Xue H., Chen L., Li Y, & Cao S. (2018). Integrated microRNA and mRNA expression profiling reveals a complex network regulating pomegranate (Punica granatum L.) seed hardness. Scientific Reports, 8, 9292.

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Pristimerin Modulates Gene Expression in H1299 Cells

Cited 1 time

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Total RNA of H1299 cells stimulated with pristimerin (0.9, 1.8, 2.7 and 3.6 μM) after 24 h was extracted using TRIzol (Thermo, Waltham, MA, USA). The PrimeScript™ RT Master Mix (Takara Biomedical Technology, Kusatsu, Japan) was used to transcribe mRNAs into cDNA. The forward and reverse primers (Takara Biomedical Technology, Kusatsu, Japan) were designed to detect the expression of integrin β1, matrix metallopeptidase 2 (MMP2) and Snail. SYBR^® Premix Ex Taq™ (TliRNaseH Plus) (Takara Biomedical Technology, Kusatsu, Japan) was utilized for RT-qPCR and GAPDH was used to normalize mRNA expression. RT-qPCR was carried out using a QuantStudio 5 (Thermo Fisher scientific, Waltham, MA, USA). The program setting for qPCR is as below: firstly, initial denaturation at 95°C for 30 sec; then 40 reaction cycles were followed by denaturation at 95°C for 5 sec, annealing at 60°C for 34 sec, and then elongation at 72°C for 30 sec 15 (link). The data were calculated using the 2^‑ΔΔCq method and GAPDH was used as control gene for normalization 16 (link). The primer sequences designed are listed in Table 1.

Li J., Guo Q., Lei X., Zhang L., Su C., Liu Y., Zhou W., Chen H., Wang H., Wang F., Yan Y, & Zhang J. (2020). Pristimerin induces apoptosis and inhibits proliferation, migration in H1299 Lung Cancer Cells. Journal of Cancer, 11(21), 6348-6355.

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Quantitative Gene Expression Analysis

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IPEC-J2 cells were harvested, and the total RNA was extracted using RNAiso Plus (Takara, Dalian, China) according to the manufacturer's instructions. The quantity and quality of the isolated RNA were determined by absorbance at 260 and 280 nm [28 (link)]. And then, cDNA was synthesized using a Reverse Transcriptase kit (Takara, Dalian, China). Briefly, quantitative PCR was performed by the QuantStudio 6 Flex Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with a total of 10 μL of assay solution containing 5 μL SYBR Green mix (Takara), 0.2 μL Rox, 3 μL deionized H₂O, 1 μL cDNA template, and 0.4 μL each of forward and reverse primers (Qingke, China). The relative gene expressions compared with the housekeeping gene β-actin were calculated by 2^-CT [29 (link)].

Lin Q., Xie K., Chen D., Yu B., Mao X., Yu J., Luo J., Zheng P., Luo Y., Yan H, & He J. (2020). Expression and Functional Characterization of a Novel Antimicrobial Peptide: Human Beta-Defensin 118. BioMed Research International, 2020, 1395304.

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Quantitative Analysis of αSMA Expression

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The mRNA expression levels of αSMA (gene: Acta2) were measured using the quantitative reverse transcription–polymerase chain reaction (qRT-PCR). HUASMCs (2.0 × 10³ cells/well) were cultured on protein-coated or uncoated surfaces for 7 days. RNA was extracted and reverse-transcribed using CellAmp™ Direct Lysis and RT (Takara Bio, Japan). Samples for qRT-PCR contained the reverse-transcribed reaction mixture (4 μl), forward and reverse primers (0.4 μM each, Takara Bio, Japan) and 1 × TB Green Premix Ex Taq II (Takara Bio, Japan). qRT-PCR was performed using a Thermal Cycler Dice^® Real Time System III (Takara Bio, Japan) with TB Green^® probes (Takara Bio, Japan) for Acta2 using the following conditions: initial hold at 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 30 s; and 1 cycle of 95°C for 15 s, 60°C for 30 s and 95°C for 15 s. The relative expression levels of target genes were calculated according to the ΔΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference. Primers were synthesized by Takara Bio, Japan. Primer sequences were as follows: Acta2, forward 5′-ATT GCC GAC CGA ATG CAG A-3′, reverse 5′-ATG GAG CCA CCG ATC CAG AC-3′; GAPDH, forward 5′-GCA CCG TCA AGG CTG AGA AC-3′, reverse 5′-TGG TGA AGA CGC CAG TGG A-3′.

Natsume K., Nakamura J., Sato K., Ohtsuki C, & Sugawara-Narutaki A. (2022). Biological properties of self-assembled nanofibers of elastin-like block polypeptides for tissue-engineered vascular grafts: platelet inhibition, endothelial cell activation and smooth muscle cell maintenance. Regenerative Biomaterials, 10, rbac111.

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Hippocampal Gene Expression Analysis

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Total RNA was isolated from the frozen hippocampus using TRIZOL reagent. cDNA was obtained using a Takara PrimeScript RT reagent kit according to the manufacturer's protocol. mRNA of NRF1, TFAM, Bax, Bcl-2, and caspase-3 was determined by the Takara TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) kit (RR820A) according to the manufacturer's instructions with the ABI7500Real-Time PCR system (Bio-Rad, CA, USA). The PCR reaction system was 25 μL (SYBR Premix Ex Taq II (Tli RNaseH Plus, 2x) 12.5 μL, Primer F (10 μM) 1 μL, Primer R (10 μM) 1 μL, cDNA 2 μL, and dH₂O 8.5 μL). The PCR conditions were as follows: 95°C for 30 seconds and 40 cycles of 95°C for 5 seconds, 60°C for 34 seconds, followed by a melting curve analysis (95°C for 15 seconds, 60°C for 1 minute, and then 95°C for 30 seconds and 60°C for 15 seconds). The sequences of the forward and reverse primers, purchased from Takara Biotechnology, used in this study are summarized in Table 1. Gene expression was normalized to β-actin and calculated by the 2-ΔΔct method. Primers were amplified with approximately equal efficiencies.

Zhang X., Zhang X., Dang Z., Su S., Li Z, & Lu D. (2020). Cognitive Protective Mechanism of Crocin Pretreatment in Rat Submitted to Acute High-Altitude Hypoxia Exposure. BioMed Research International, 2020, 3409679.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was isolated from each human sample using 1 mL RNAiso Plus reagent (TaKaRa, Otsu, Japan), as described previously (Zhang et al., 2022 (link)). RNA concentration and quality were estimated at 260/280 nm using an Ocean Optics spectrophotometer (Dunedin, United States). RNA was reverse-transcribed into cDNA using oligo (dT) primers. Next, the PCR mixture consisting of TB Green (10 uL), forward and reverse primers (10 uL, TAKARA, Japan), DNA (1 uL) and ddH2O (7.0 uL) were placed in the Realtime System (CFX Connect, United States). The reaction parameters were 95°C for 5 min (1 cycle), followed by 40 cycles of 95°C for 30°s, and 60°C for 30 s. The relative expression of mRNA was quantified using the 2^−ΔΔCT method.

Yang X., Zhang X., Shen K., Wang Z., Liu G., Huang K., He Z., Li Y., Hou Z., Lv S., Zhang C., Yang H., Liu S, & Ke Y. (2023). Cuproptosis-related genes signature and validation of differential expression and the potential targeting drugs in temporal lobe epilepsy. Frontiers in Pharmacology, 14, 1033859.

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Quantitative PCR Analysis of Bougainvillea

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The specific qPCR primers were designed using the PRIMER5.0 software and synthesized by Sangon Biotech Co., Ltd., Shanghai, China (shown in Table S1). The Q8W4U4, Q5J7U5, A0A0D3RWN7, and Q09EH2 sequences were downloaded from NCBI and abbreviated as Q8, Q5, A0A, and Q09, respectively. Bougainvillea glabra 18s rRNA served as the internal control.
PCR was performed in a final volume of 20 µL, which contained 50 ng of cDNA, 10 µL of 2×TB Green Premix Ex Taq (Tli RNaseH Plus), 0.4 µL of ROX Reference dye II, and 2 pmol each of the forward and reverse primers (Takara, Dalian, China). The PCR programs were conducted at 95 °C for 20 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Ct values were calculated based on technical triplicate experiments performed on three biological replicates. The mRNA levels were quantified using the ∆Ct method. A melting curve analysis was performed to confirm the presence of a single PCR product after each reaction.

Lin Y., Xu L., Li Y., Wu X., Liu Y., Zhu H, & Zhou H. (2021). Ribosome-Inactivating Proteins of Bougainvillea glabra Uncovered Polymorphism and Active Site Divergence. Toxins, 13(5), 331.

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