Pups were sacrificed after prior anesthesia 48 h after HI injury (n=6/group) and the cortical tissue in the peri-infarct was obtained. Total RNA was extracted from cerebral cortices and BV2 cells using Trizol Reagent (Invitrogen, Grand Island, NY, USA) and reverse transcribed to cDNA using a cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, WA, USA). RT-qPCR was performed by the SYBR Green method (K1622, Applied Biosystems, CA, USA). The reaction was performed at 95 ℃ for 5 min followed by 40 cycles of 95 ℃ for 15 s and 55 ℃ for 40 s on an IQ5 PCR machine (Bio-Rad, Hercules, CA, USA). The relative expression of the targeted mRNA was normalized to the expression of β-actin. Sequence-specific primers were designed according to previous literature (20 (link)).
Iq5 pcr machine
Manufactured by Bio-Rad
Sourced in United States
The IQ5 PCR machine is a real-time PCR instrument designed for quantitative analysis of nucleic acid samples. It features a compact design, a user-friendly interface, and the ability to perform precise temperature control and data acquisition for various real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr primescript rt pcr kit, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Nucleospin rna 2 kit, by Macherey-Nagel (1 mentions)
Nucleospin extract 2 kit, by Macherey-Nagel (1 mentions)
Sensifast sybr fluorescein kit, by Meridian Bioscience (1 mentions)
Nuclease free water, by Promega (1 mentions)
Oligo dt primer, by Promega (1 mentions)
Evolution 500, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Rnasin, by Promega (1 mentions)
Dntp mix, by Promega (1 mentions)
Total rna extraction kit, by Corning (1 mentions)
M mlv reverse transcription kit, by Promega (1 mentions)
7 protocols using iq5 pcr machine
1
Quantitative Analysis of mRNA Expression
2
Real-Time PCR Quantification of Gene Expression
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We carried out a real-time PCR using a SYBR® PrimeScript™ RT-PCR Kit (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. Total RNA was reverse-transcribed to obtain cDNA using oligo-dT and reverse transcriptase at 50°C for 30 min and then heat inactivation of the enzyme at 85°C for 5 min. The cDNA samples were then mixed with SYBR® dye. We carried out gene-specific and quantitative real-time PCR using a Bio-Rad iQ5 PCR machine (Bio-Rad, USA). The PCR program consisted of pre-denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 10 s, annealing at 55°C or 56°C (for β-actin and amylase, respectively) for 15 s and extension at 72°C for 12 s.The PCR was then heated from 60 to 95°C to produce the melting curve. One negative control was included in all reactions.
3
Quantification of TRβ1 mRNA Expression
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Total RNA from tissues was extracted using Trizol solution. Reverse transcription (RT) was performed in a 20 μL reaction system according to the manufacturer’s recommendation. To analyze TRβ1 mRNA expression, real-time quantitative RT-polymerase chain reaction (RT-PCR) was performed. Briefly, RT-PCR amplification was carried out for each sample in a 12.5 μL final reaction mixture containing 1 μL of cDNA, 3 nM TaqMan probe, 5.5 mM MgCl2, 10 nM of each primer, 0.6 U of Platinum Taq polymerase, and 200 μM of deoxyguanosine triphosphate. After an initial denaturation step at 95°C for 1.5 minutes, 40 cycles of 15 seconds at 95°C and 56 seconds at 60°C for annealing and extension were run on an iQ5 PCR machine (Bio-Rad Laboratories, Hercules, CA, USA).
TRβ1 mRNA levels were normalized to β-actin. The primer sequences used in the present study are presented inTable 1 . All reactions were performed in duplicate. The relative expression of TRβ1 was analyzed by the comparative Ct method. Thermal dissociation plots were examined for biphasic melting curves.
TRβ1 mRNA levels were normalized to β-actin. The primer sequences used in the present study are presented in
4
RNA Isolation and RT-PCR Analysis
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After surgery, the mice were fed the WTD for another 15 weeks. Subsequently, mice were sacrificed and organs were collected and peritoneal leukocytes isolated by lavage of the peritoneum with ice-cold PBS. Total RNA from spleen, liver and peritoneal leukocytes was isolated using the Nucleospin RNA II kit (Macherey-Nagel, Düren, Germany) according to manufacturer's instructions. One microgram of total RNA was converted to cDNA with iScript cDNA Synthesis kit (Biorad) and purified with Nucleospin Extract II kit (Macherey-Nagel). Realtime polymerase chain reaction (RT-PCR) was conducted on the IQ5 PCR machine (Biorad) using the Sensimix SYBR Green RT-PCR mix (Quantace, London, UK). mRNA levels were normalized to mRNA levels of β2 microglobulin, cyclophilin, and acidic ribosomal phosphoprotein P0 (36B4).
5
RNA Isolation and qPCR Analysis
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The total RNA was isolated from extracted cells using the RNeasy mini kit (Qiagen, Germantown, MD) and quantified with a ultraviolet spectrophotometer (Evolution 500, Thermo Fisher Scientific, Grand Island, NY).
The first strand of complementary DNA was synthesized from 1 mg of RNA with oligo-dT primers, M-MLV reverse transcriptase, dNTP mix, RNasin, and nucleasefree water (all reagents from Promega, Madison, WI) according to the manufacturer's instructions. Quantitative polymerase chain reaction (PCR) was carried out using the SensiFAST SYBR Fluorescein Kit (Bioline, Singapore). The PCR amplification and measurement were conducted in an iQ5 PCR machine (Bio-rad, Singapore) for 2 minutes of denaturation at 958C, and 40 cycles of denaturation at 958C for 10 seconds, and annealing/extension at 608C for 30 seconds. All gene primers used in this study are listed in Table 2.
The first strand of complementary DNA was synthesized from 1 mg of RNA with oligo-dT primers, M-MLV reverse transcriptase, dNTP mix, RNasin, and nucleasefree water (all reagents from Promega, Madison, WI) according to the manufacturer's instructions. Quantitative polymerase chain reaction (PCR) was carried out using the SensiFAST SYBR Fluorescein Kit (Bioline, Singapore). The PCR amplification and measurement were conducted in an iQ5 PCR machine (Bio-rad, Singapore) for 2 minutes of denaturation at 958C, and 40 cycles of denaturation at 958C for 10 seconds, and annealing/extension at 608C for 30 seconds. All gene primers used in this study are listed in Table 2.
6
Real-Time PCR for Gene Expression Analysis
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We performed a real-time PCR using a SYBR ® PrimeScript TM RT-PCR Kit (Takara Biotechnology, Dalian, China) following the manufacturer instructions. Total RNA was reversetranscribed to yield cDNA using oligo-dT and reverse transcriptase at 50°C for 30 min, which was followed by heat inactivation at 85°C for 5 min. The cDNA samples were then mixed with SYBR ® dye. We performed gene-specific and quantitative real-time PCR using a Bio-Rad iQ5 PCR machine (Bio-Rad, USA). The PCR program consisted of predenaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 10 s, annealing extension at 55°C or 56°C (for β-actin and amylase, respectively) for 15 s, and at 72°C for 12 s, followed by heating of the product from 60 to 95°C to generate the melting curve for each PCR product. One negative control was included in all of the reactions.
7
Chicken Skin RNA Extraction and RT-qPCR
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Total RNA was extracted from chicken breast skin using a total RNA extraction kit (Axygen, USA). The quality of RNA was evaluated on 1.5% agarose gel electrophoresis and using BioSpec-nano spectrophotometer (Shimadzu, Japan). First strand cDNA was synthesized from 2 µg total RNA with the M-MLV reverse transcription kit (Promega, USA) and stored at -20°C.
Primer characteristics are shown in Table 2. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). PCR amplification was carried on a Bio-Rad IQ5 PCR machine. The 12.5-µL PCR included: cDNA, 1.0 µL; pH 8.0 SDW, 4.75 µL; SYBR Green I Premix, 6.25 µL; 10 µM Forward primer, 0.25 µL; 10 µM Reverse primer, 0.25 µL. The PCR was carried out at 95°C for 1 min, 95°C for 15 s, and 63°C for 25 s, for a total of 40 cycles; melting curves were generated at 55 to 90°C.
Primer characteristics are shown in Table 2. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). PCR amplification was carried on a Bio-Rad IQ5 PCR machine. The 12.5-µL PCR included: cDNA, 1.0 µL; pH 8.0 SDW, 4.75 µL; SYBR Green I Premix, 6.25 µL; 10 µM Forward primer, 0.25 µL; 10 µM Reverse primer, 0.25 µL. The PCR was carried out at 95°C for 1 min, 95°C for 15 s, and 63°C for 25 s, for a total of 40 cycles; melting curves were generated at 55 to 90°C.
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