ABCC4 mRNA levels in the cells were quantitatively measured using GoTaq® qPCR Master Mix, 2× (Promega KK., Tokyo, Japan) and gene-specific primers for ABCC4 [forward primer (Takara Bio Inc.; 5095-085), reverse primer (Takara Bio Inc.; 5095-086)] and GAPDH (housekeeping gene), which was used as the internal control [forward primer (Takara Bio Inc.; 10000459), reverse primer (Takara Bio Inc.; 20000459)] by using Applied Biosystems 7500Fast Real-Time PCR System (Thermo Fisher Scientific Inc.). Concretely, the RNA quality used for qPCR was 1.50–1.70. As a reaction solution (20 μL), GoTaq® qPCR Master Mix, 2 × (10 μL), 50 μM forward primer (0.08 μL), 50 μM reverse primer (0.08 μL), nuclease-free water (5.84 μL) and 2.5 ng/μL template cDNA (4 μL) were mixed each well in a 96-well plate. The Quantitative Real-Time PCR reaction consisted for 2 min at 95 °C, 40 cycles of reactions for 15 s at 95 °C, for 1 min at 60 °C, as a dissociation stage, for 15 s at 95 °C, for 1 min at 60 °C and for 15 s at 95 °C.
Reverse primer
Manufactured by Takara Bio
Sourced in Japan
A reverse primer is a short DNA sequence used in reverse transcription and polymerase chain reaction (PCR) to initiate the synthesis of complementary DNA (cDNA) from an RNA template. It binds to the 3' end of the target RNA sequence and enables the reverse transcriptase enzyme to generate a cDNA strand.
Automatically generated - may contain errors
Lab products found in correlation
Forward primer, by Takara Bio (4 mentions)
Applied biosystems 7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Gotaq qpcr master mix 2, by Promega (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Deoxynucleotide triphosphate, by Takara Bio (1 mentions)
Primer premier 5, by Premier Biosoft (1 mentions)
Rtaq dna polymerase, by Takara Bio (1 mentions)
Miseq 300pe, by Illumina (1 mentions)
Taq buffer, by Takara Bio (1 mentions)
Ex taq polymerase, by Takara Bio (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
6 protocols using reverse primer
1
Quantifying ABCC4 mRNA Expression
2
Amplification and detection of UPF1 gene
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DNA was extracted from 293T cells following transfection with the UPF1 lentiviral vector. The UPF1 gene was amplified using primers designed by Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, CA, USA) and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The primer sequences were as follows: UPF1 forward, 5′-AACACTATCAACCCGGGAGC-3′ and reverse, 5′-GCTAGCTCATTTGTCGTCATC-3′; GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. The PCR reaction system contained 13.25 µl double-distilled water, 5 µl 10X PCR buffer (Mg2+ Plus), 2 µl deoxynucleotide triphosphates, 2 µl DNA template, 1 µl forward primer, 1 µl reverse primer and 0.25 µl rTaq DNA polymerase (all from Takara Bio, Inc., Otsu, Japan), in a total volume of 25 µl. The PCR thermocycling conditions were as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 1 min; and 72°C for 10 min. The PCR products were separated by 1.5% agarose gel electrophoresis. The positive control was GAPDH.
3
Rumen Microbiome DNA Extraction and 16S Amplification
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Total genomic DNA was extracted from the rumen fluids using TIANamp DNA Isolation Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. The DNA was quantified on 2% agarose gel and by using nanodrop (Thermo Scientific). Archaeal and bacterial 16S rRNA genes V3-V4 region (from 349 to 806) were amplified from extracted DNA using barcoded primers Arch349F (5’-GYGCASCAGKCGMGAAW-3’) and Arch806R (5'-GGACTACVSGGGTATCTAAT-3') [16 (link)]. Each 50 μL PCR reaction contained 10 ng DNA, 39 μL Molecular Biology Grade water, 5 μL 10X Ex Taq Buffer with 5 μL dNTPs (2.5 mM each), 0.5 μL Forward Primer (50 pM), 0.5 μL Reverse Primer (50 pM), and 0.25 μL Ex Taq Polymerase (5U/μL) (Takara Bio Inc. Japan). PCR was performed under following conditions: 94°C for 3 min; followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 5 min; followed by a final extension at 72°C for 5 min. Three replicates of PCR were performed for each sample. These PCR products were pooled, purified and then quantified by using nanodrop (Thermo Scientific). Then, next-generation sequencing was performed by Illumina MiSeq 300PE which was conducted by Macrogen Inc. (Seoul, South Korea).
4
Reverse Transcription RNA Analysis
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The reverse‐transcription mixture included 2 μL of PrimeScript Two‐Step Enzyme Mix (Takara, Tokyo, Japan), 15 μL of 2 × 1 Step Buffer (Dye Plus), 1 μL of forward primer (100 μm ), 1 μL of reverse primer (100 μm ), 3 μL of random primers at 100 μm (Takara), 1 μL (500 ng) of total RNA, and 7 μL of RNase‐free ddH2O in a final volume of 30 μL. The mixture was incubated at 72 °C for 15 min and 98 °C for 5 s in a 7279 Thermocycler (Applied Biosystems, Foster City, CA, USA).
5
Quantitative mRNA Analysis of ABCC4
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ABCC4 mRNA levels in the cells were measured quantitatively using GoTaq® qPCR Master Mix, 2× (Promega, Tokyo, Japan) and gene-specific primers, for ABCC4 (forward primer, 5095-085 (Takara Bio), reverse primer, 5095-086 (Takara Bio)) and the housekeeping gene GAPDH as an internal control (forward primer, 10000459 (Takara Bio), reverse primer, 20000459 (Takara Bio)) with an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA).
6
Quantification of Renal Gene Expression
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Mouse embryonic kidneys were harvested between E11.5 and E14.5, and tissues were collected for culturing. Total RNA was extracted using the RNAiso Plus Reagent (cat. no. 9108; Takara Bio, Inc., Tokyo, Japan). A total of 500 ng RNA was reverse transcribed into first strand cDNA using the Primescript RT reagent kit (cat. no. DRR037A; Takara Bio, Inc.). SYBR Premix Ex Taq (10 µl; cat. no. DRR041A; Takara Bio, Inc.) was added to the qPCR reaction mixture, including 0.4 µl forward primer (10 µM; Takara Bio Inc.), 0.4 µl reverse primer (10 µM; Takara Bio Inc.), 2 µl cDNA and 7.2 µl distilled H2O, at a final volume of 20 µl. The primers used to detect SHH, Fgf8 and Fgf10 mRNA expression levels are listed in Table I . Thermal cycling was performed using the Roche LightCycler 480 system (Roche, Basel, Switzerland). The mRNA expression levels of each target gene were determined using a calibration curve of standards, and expressed relative to GAPDH expression levels.
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