RNA was extracted from cells using TRIzol (Invitrogen) as recommended by the manufacturer, and the concentration and quality were measured by a NanoDrop 2000 (ThermoFisher, Waltham, USA) [54 (link)]. Complementary DNA (cDNA) was synthesized with a reverse transcription kit (Takara, Tokyo, Japan). Then, Mix (Toyobo, Japan) and specific primers for every gene were used to perform RT‒qPCR on a Real-time System (Roche, Basel, Switzerland) (Additional file 8 : Table S4). The expression levels of genes were normalized to ribosomal protein S20 (RPS20) to obtain the relative expression using the 2–ΔΔCt method according to a previous study [12 (link), 56 (link)].
Real time system
Manufactured by Roche
Sourced in Switzerland
The Real-time System is a laboratory instrument designed for real-time monitoring and analysis of various biological samples. It utilizes advanced detection technologies to provide accurate and reliable data in a timely manner. The core function of this system is to enable real-time measurement and quantification of specific analytes or targets within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Reverse transcription system, by Promega (1 mentions)
Nucleozol reagent, by Macherey-Nagel (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Sybr green master mix, by Roche (1 mentions)
7 protocols using real time system
1
RNA Extraction and RT-qPCR Analysis
2
Quantitative Real-Time PCR Gene Expression Analysis
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Revert Aid First Strand cDNA Synthesis Kit (Fermentase) was utilized for synthesizing cDNA. Quantitative Real-Time PCR (qPCR) experiments were implemented and replicated three times using BIO FACT real-time master mix. Primers were designed by Primer 3 online software [42 (link)] and synthesized by Gene Script Company. Primer’s properties were summarized in the S2 Table . The primer specificity was confirmed using Primer blast in NCBI (https://www.ncbi.nlm.nih.gov ). The qRT-PCR reaction was made with 5μl of diluted cDNA, 10 μl of 2X PCR Master Mix, and 1 μl of each primer (10 pmol). The final volume reached 20 μl with double distilled water. The PCR program consisted of several steps: 5 min at 94˚C, 40 cycles of 10 sec at 95 ˚C, 60 sec at 49 ˚C, 10 sec at 72 ˚C, and a final step of 30 sec at 72 ˚C. A melting curve analysis including 81 cycles at 55–95 ˚C with 0.5˚C increases in each cycle was performed to evaluate the primer specificity. The real-time PCR was done using the Roche Real-time system. Relative expression ratios were calculated through the comparative ΔΔCT method by REST software for relative gene quantification, according to equation 1. A housekeeping gene (in this study, Actin) was applied as a reference gene with equal transcripts in all stages and tissues.
3
Gene Expression Analysis of Embryonic Hippocampi
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Hippocampi from E17.5 embryos were collected according to the above-mentioned method. Tissues were homogenized and lysed in TRIzol reagent according to the manufacturer’s protocol (Life Technology). RNA yields were measured using a NanoDrop 2000 (Thermo, Waltham, MA, USA). A reverse transcription system (Promega) was used to obtain cDNA. Real-time PCR was performed on an Applied Biosystems real-time system according to the detailed instructions provided by FastStart Universal SYBR Green Master (Roche, Basel, Switzerland). For normalization of gene expression, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Primers used are listed in Table 1
4
Quantifying Gene Expression in Cultured MSCs
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Total RNA was extracted from cultured MSCs using NucleoZOL reagent (MACHEREY‑NAGEL) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using the PrimeScript RT Reagent Kit (TaKaRa). Real-time RT‒qPCR was carried out using a Roche Real-time System. Ppia was amplified at the same time to normalize gene expression. Each experiment was repeated three times to determine differences in relative gene expression. The sequences of the PCR primers used in this study are shown in Table S1 .
5
Quantitative RNA Expression Analysis
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Total RNA was isolated from J774A.1 cells or foot pad tissue with TRIzol Reagent (TIANGEN, China), and cDNA was synthesized with a reverse transcriptase kit. A real-time system (Roche, USA) was used for PCR with SYBR Green Master Mix. The relative amounts of the target mRNA were normalized to the expression level of the reference gene GAPDH, and the data were analyzed through the 2−ΔΔCt method. The primer sequences are shown in Table 1
6
Quantitative Real-Time PCR Analysis of Glycolytic Genes in Mouse Blood
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Total RNA was extracted from the peripheral blood of mice using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, United States). Reverse transcription of total RNA was performed using a high-capacity cDNA reverse transcription kit (code: FSQ-101, Osaka, Japan). The primer sequences for qRT-PCR are as follows for G6pd primers: 5′-CAG GGA CGA GCT CCT TGA G-3′ and 5′-GGG GGT TCA CCC ACT TGT AG-3′; Eno3 primers: 5′-CGC AGA TCT TGC AGG CAA TC-3′ and 5′-GGG TCA TCG GGT GAC TTG AA-3′; Taldo1 primers: 5′-AAA AAG TTG GCA TGT CGA GC-3′ and 5′-GCT GAT CCC AGC TTC CTT GT-3′; Pfkl primers: 5′-TCA TGT GTG TCA TCC CAG CC-3′ and 5′-CAT GCG GTG CTC GAA ATC AG-3′; Pgd primers: 5′-CCA TGG CCC AAG CTG ACA TC-3′ and 5′-ACC GTC TTG TGG TGT CCC TA-3′. For qRT-PCR, SYBR Green PCR Master Mix (Cat. No. 04913850001, Roche, Germany) and the BIO-GENER Real-Time System (China) were used according to the manufacturer's instructions. The number of mice in each group was eight, and each sample was tested in triplicate-independent qRT-PCR. The threshold cycle (Ct) values were standardized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) values measured on the same plate, and the 2−ΔΔCt method ·was used to determine the fold changes in gene expression [33 (link)].
7
Quantitative Analysis of miR-92b-3p and Genes
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The RNA was extracted from cells using Trizol (Invitrogen) as recommended by the manufacturer and the concentration and quality were measured by the NanoDrop 2000 (ThermoFisher, Waltham, USA). The complementary DNA (cDNA) was synthesized with a reverse transcription kit (Takara, Tokyo, Japan). Then, the Mix (Toyobo, Japan) and specific primers for every gene (Table S1 ) were used to perform RT-qPCR on a Real-time System (Roche, Basel, Switzerland). The expression levels of miR-92b-3p and genes were normalized with U6 and RPS20 to obtain the relative expression using the 2–ΔΔCt method, respectively.
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