Q5 instrument

Manufactured by Bio-Rad

Sourced in United States, Japan

The Bio-Rad Q5 instrument is a real-time PCR system designed for sensitive and accurate nucleic acid quantification. It features a compact design, a high-resolution touch screen interface, and supports a range of sample volumes and reaction formats. The Q5 instrument is capable of performing real-time PCR experiments to quantify DNA, RNA, and other nucleic acid targets.

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Lab products found in correlation

Revertra ace qpcr rt kit, by Toyobo (3 mentions) Sybr qpcr mix, by Toyobo (2 mentions) Qpcr rt kit, by Toyobo (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Thunderbird sybr qpcr mix, by Toyobo (1 mentions) E z n a total rna kit 1, by Omega Bio-Tek (1 mentions) Reverse transcription kit, by Toyobo (1 mentions) First strand cdna synthesis kit, by Toyobo (1 mentions) Sybr green realtime pcr master mix, by Toyobo (1 mentions) Total rna extraction kit, by Toyobo (1 mentions)

5 protocols using q5 instrument

Quantitative gene expression analysis

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Total RNA was extracted using E.Z.N.A Total RNA extracting kit (Omega, United States), and reverse transcription was performed using ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan) based on the measured total RNA concentration. Reverse transcription and quantitative PCR (RT-qPCR) was conducted by RT-qPCR kit (Toyobo) and Bio-Rad Q5 instrument (Bio Rad). The primers sequences involved were as follows (F, forward; R, reverse):
GAPDH, F 5′-ACCCAGAAGACTGTGGATGG-3′ and R 5′-CACATTGGGGGTAG GAACAC-3′; NFATc1, F 5′-GACCCGGAGTTCGACTTCG-3′ and R 5′-TGACACT AGGGGACACATAACTG-3′; TRAP, F 5′-CACTCCCACCCTGAGATTTGT-3′ and R 5′-CATCGTCTGCACGGTTCTG-3′; Cathepsin K, F 5′-GAAGAAGACTCACCA GAAGCAG-3′ and R 5′-TCCAGGTTATGGGCAGAGATT-3′; MMP9, F 5′-CTGGA CAGCCAGACACTAAAG-3′ and R 5′-CTCGCGGCAAGTCTTCAGAG-3′.

Guo J., Ren R., Sun K., Yao X., Lin J., Wang G., Guo Z., Xu T, & Guo F. (2020). PERK controls bone homeostasis through the regulation of osteoclast differentiation and function. Cell Death & Disease, 11(10), 847.

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Osteoclast Differentiation Assay with Iguratimod

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BMMCs were seeded at a density of 1×10⁵ cells/mm² in 6-well plates. Cells were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) in the presence of vehicle or 3 µg/ml iguratimod for 5 days. Then total RNA was extracted from BMMCs using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) as previously described (21 (link)). First-strand cDNA was synthesized using ReverTra Ace qPCR RT kit (Toyobo Co., Ltd., Osaka, Japan) to perform RT-qPCR using the Thunderbird SYBR qPCR Mix (Toyobo Co., Ltd.) and a Bio-Rad Q5 instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All reactions were performed according to the manufacturer's instructions, and target gene expression was normalized to the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative expression levels of each gene were calculated using the comparative 2^−ΔΔCt method (22 (link)). The primers used for RT-qPCR are listed in Table I.

Wu Y.X., Sun Y., Ye Y.P., Zhang P., Guo J.C., Huang J.M., Jing X.Z., Xiang W., Yu S.Y, & Guo F.J. (2017). Iguratimod prevents ovariectomy-induced bone loss and suppresses osteoclastogenesis via inhibition of peroxisome proliferator-activated receptor-γ. Molecular Medicine Reports, 16(6), 8200-8208.

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Quantitative PCR Analysis of Osteoclastogenesis

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Quantitative real-time reverse transcription PCR was carried out as described before (Zhang et al., 2015 (link)). BMMs were planted into a 6-well plate (1 × 10⁵ cells/well), cultured with the medium containing RANKL (100 ng/mL) and M-CSF (30 ng/mL), treated with or without ulinastatin (800 U/mL). The extraction of total RNA was conducted through E.Z.N.A Total RNA Kit I (Omega Bio-Tek, Norcross, GA, United States). Reverse transcription was conducted by using Rever Tra Ace qPCR RT Kit (Toyobo, Osaka, Japan), qRT-PCR was conducted by using SYBR qPCR Mix (Toyobo, Osaka, Japan) on Bio Rad Q5 instrument (Bio-Rad Laboratories, CA, United States), and the target genes’ expression was standardized to the reference gene GAPDH. All procedures were conducted following manufacturer’s protocol. Primers used for qRT-PCR (F represents forward; R represents reverse) : GAPDH (F) 5′-CTCCCACTCTTCCACCTTCG-3′, (R) 5′-TTGCTGTAGCCGTATTCATT-3′; TRAP (F) 5′-TACCTGTGTGGACATGACC-3′, (R) 5′-CAGATCCATAGTGAAACCGC-3′; Cathepsin K (F) 5′-TGTATAACGCCACGGCAAA-3′, (R) 5′-GGTTCACATTATCACGGTCACA-3′; NFATc1 (F) 5′-CAACGCCCTGACCACCGATAG-3′, (R) 5′-GGGAAGTCAGAAGTGGGTGGA-3′; RANK (F) 5′-CAGGAGAGGCATTATGAGCA-3′, (R) 5′-GGTACTTTCCTGGTTCGCAT-3′; uPAR (F) 5′-AACTCAGCCTCATTGCCTCT-3′, (R) 5′-TCCTCAAAGATGGAGCAGGG-3′.

Huang J.M., Ren R.Y., Bao Y., Guo J.C., Xiang W., Jing X.Z., Shi J., Zhang G.X., Li L., Tian Y., Kang H, & Guo F.J. (2018). Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR. Frontiers in Pharmacology, 9, 1016.

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Quantitative Real-Time RT-PCR Analysis

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Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was carried out as mentioned previously [67 (link)]. After being seeded, cultured, and treated, the cells were applied for extracting total RNA by using E.Z.N.A Total RNA Extraction Kit (OMEGA Bio-Tek, USA), and Reverse Transcription Kit (Toyobo, Japan) was used to reverse transcribe RNA to cDNA, qRT-PCR procedures were subsequently performed on Bio-Rad Q5 instrument (Bio-Rad Laboratories, USA) using SYBR qPCR Mix (Toyobo, Japan).
The primers were synthesized by Tsingke Biotechnology (Beijing, China), and the sequences (5′ to 3’) are shown in Table 1.Table 1

Primers sequences used for qRT-PCR.

Table 1

Gene	Forward	Reverse
GAPDH	GGCACAGTCAAGGCTGAGAATG	ATGGTGGTGAAGACGCCAGTA
SDF-1	ATGAACGCCAAGGTCGTGGTC	TGGCTGTTGTGCTTACTTGTTT
VEGF	TCACCAAAGCCAGCACATAG	TTTCTCCGCTCTGAACAAGG
Tie2	CGAGGTCAAGAAGTGTATGT	TGATGTATGGTCCTATGGTGT
RUNX2	AACCACAGAACCACAAGTGCG	AAATGACTCGGTTGGTCTCGG
ALP	GGGACTGGTACTCGGACAAT	GGCCTTCTCATCCAGTTCAT
OCN	CTGACCTCACAGATCCCAAGC	TGGTCTGATAGCTCGTCACAAG
OPN	GGGGACTATGCACCTGAGC	GACTGTCGAAATGGGCTACCT

Ren R., Guo J., Song H., Wei Y., Luo C., Zhang Y., Chen L., Gao B., Fu J, & Xiong W. (2023). A novel implant surface modification mode of Fe3O4-containing TiO2 nanorods with sinusoidal electromagnetic field for osteoblastogenesis and angiogenesis. Materials Today Bio, 19, 100590.

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Quantitative Real-Time PCR for Nrf2 Expression

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Total mRNA expression was extracted with the total RNA extraction kit (Toyobo, Japan) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from total RNA with first-strand cDNA Synthesis Kit (Toyobo, Japan). Finally, cDNA was amplified using SYBR Green Real-time PCR Master Mix (Toyobo, Japan) with the following cycling conditions: 30 s of polymerase activation at 95 °C, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The intensity of fluorescent was measured by Bio-Rad Q5 instrument (Bio-Rad Laboratories, CA). GAPDH was selected as the internal control. Sequences of primers used were listed as follows: Nrf2: forward (TAGATGACCATGAGTCGCTT), reverse (CTGTAACTCGGGAATGGAAA); GAPDH: forward (AACATCAAATGGGGTGAGGCC), reverse (GTTGTCATGGATGACCTTGGC).

Yao X., Yu S., Jing X., Guo J., Sun K., Guo F, & Ye Y. (2020). PTEN inhibitor VO-OHpic attenuates GC-associated endothelial progenitor cell dysfunction and osteonecrosis of the femoral head via activating Nrf2 signaling and inhibiting mitochondrial apoptosis pathway. Stem Cell Research & Therapy, 11, 140.

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