The differentially expressed miRNAs selected from the microarray were validated by RT-qPCR analysis. cDNA was generated with the PrimeScript™ RT-qPCR kit (Takara Bio, Inc., Otsu, Japan) in accordance with the manufacturer's instructions. PCR was performed in a 7900HT fast RT-qPCR instrument (Applied Biosystems Life Technologies, Singapore). The primers used were purchased from Ribobio (Guangzhou, China) and the sequences for the control were as follows: U6, forward 5'-GCGCGTCGTGAAGCGTTC -3' and reverse 5'-GTGCAGGGTCCGAGGT-3'.All reactions involved initial denaturation at 95˚C for 3 min, followed by 40 cycles of 95˚C for 10 sec, 60˚C for 20 sec and 70˚C for 1 sec. miRNA quantification was performed by using the miDETECT A TrackTM miRNA qRT-PCR Starter Kit (Ribobio) in a 7900HT fast RT-qPCR instrument (Applied Biosystems, Foster City, CA, USA). All quantitative reactions, including the no-template controls, were run in duplicate. The small nuclear RNA U6 was used as the normalization control.
7900ht fast rt qpcr instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7900HT Fast Real-Time PCR System is a high-performance instrument for quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis. It features advanced optics and thermal cycling technology to enable rapid and precise gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Primescript rt qpcr kit, by Takara Bio (2 mentions)
Primescript rt kit, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Midetect a track mirna qrt pcr starter kit, by RiboBio (1 mentions)
6 protocols using 7900ht fast rt qpcr instrument
1
Validating Differentially Expressed miRNAs
2
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, the selected results from RNA-sequencing (RNA-seq) were verified by RT-qPCR analysis. cDNA was produced using a Prime Script™ RT-qPCR kit (Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was performed using SYBR® Premix Ex Taq™ (Takara Bio, Inc.) on a 7900HT fast RT-qPCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) with technical triplicates. The gene-specific primers used for RT-qPCR are listed in Table I . Thermocycling conditions were as follows: Initial denaturation at 95˚C for 5 min, followed with 35 cycles at 95˚C for 30 sec and 60˚C for 30 sec. The relative gene expression levels were determined using the 2-ΔΔCq method (31 (link)).
3
Quantitative PCR Gene Expression Analysis
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The complementary DNA was produced by the Prime Script™ RT‐qPCR Kit (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. The quantification PCR were carried out in 7900HT fast RT‐qPCR instrument (Applied Biosystems, Life Technologies, Cologne, Germany) with SYBR® Premix Ex Taq™ II (Takara Bio Inc., Tokyo, Shiga, Japan). The endogenous control was the expression of glyceraldehyde‐3‐phosphate dehydrogenase was used as the control. The level of genes were calculated by the 2 − Δ Δ C t
4
Quantifying Gene Expression by RT-qPCR
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Total RNA was extracted using an RNA Isolation kit according to the manufacturer’s instructions. A NanoDrop™ ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, MA, USA) was used to determine the quantity and purity of RNA. Expression of complementary DNA was measured using a Prime Script™ RT kit (TaKaRa Biotechnology, Otsu, Japan). qPCR was undertaken on a 7900HT fast RT qPCR instrument (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions. Actin RNA was used as an internal control. The expression level was normalized to the average value in the control group to obtain the relative level.
5
CRISPR Lentivirus Infection Alters Hippocampal mRNA
6
RNA Extraction and qPCR Analysis
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An RNA Isolation kit was applied to extract total RNA according to manufacturer instructions. A NanoDrop™ ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, MA, USA) was used to determine the quantity and purity of RNA. Expression of complimentary DNA was measured using a Prime Script™ RT kit (TaKaRa Biotechnology, Otsu, Japan). qPCR was undertaken on a 7900HT fast RT qPCR instrument (Applied Biosystems, Foster City, CA, USA) according to manufacturer instructions. The RNA of actin was used as an internal loading control.
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