Myocardial gene expression of Sfrp2 was performed by quantitative real-time PCR (qRT-PCR). Total RNA was extracted using the TRIzol reagent (Life Technologies). The RNA samples reverse-transcribed to cDNA using an RNA reverse were transcribed with Maxima First Strand cDNA Synthesis Kit (Takara, Dalian, China). The primers used were as follows: forward 5′-CATGGGACAGAAACAGGGTGGA-3′ and reverse 5′-GAGGTCGCAGAGTGGAAGTGGT-3′ for Sfrp2; forward 5′-GTATCGGACGCCTGGTTAC-3′ and reverse 5′-ACTGGAACATGTAGACCATGTAGTT-3′ for GAPDH.
Maxima first strand cdna synthesis kit
Manufactured by Takara Bio
Sourced in Japan
The Maxima First Strand cDNA Synthesis Kit is a laboratory tool designed for the reverse transcription of RNA into complementary DNA (cDNA). The kit includes the necessary reagents and enzymes to perform this process efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Cell total rna isolation kit, by Foregene (1 mentions)
S1000 pcr instrument, by Bio-Rad (1 mentions)
Maxima sybr green qpcr master mix, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Takara Bio (1 mentions)
Lightcycler 480 2 system, by Roche (1 mentions)
Qiazol, by Qiagen (1 mentions)
Qiazol reagent, by Qiagen (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
6 protocols using maxima first strand cdna synthesis kit
1
Quantifying Myocardial Sfrp2 Expression
2
Evaluating Key lncRNA and mRNA Levels in Ovarian Cancer
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The expression levels of two key lncRNAs (HAND2-AS1 and LEMD1-AS1) and five key mRNAs (MPPED2, SAMD12, LONRF1, SORD and SERPINA1) were assessed in the control cell line (IOSE80) and the three OC cell lines (A2780, SK-O-V3 and OVCAR-3) to verify the reliability of the present results. Total RNA was extracted from the cell lines using a Cell Total RNA Isolation Kit (Foregene). Random primers and a Maxima First Strand cDNA Synthesis Kit (Takara Bio, Inc.) were used to synthesize cDNA. SYBR® Green Master Mix was used for qPCR. The primers, designed and synthesized by RiboBio, are listed in Table SI . GAPDH was used as the internal control. The relative expression was calculated using the 2−ΔΔCq method (24 (link)). All experiments were performed three times.
3
Quantification of mRNA and miRNA Levels
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Total RNA was isolated from cell lines, patient samples with Trizol reagent (Invitrogen). After treatment with DNase I, RNA was reverse transcribed into cDNA with Thermo scientific maxima first strand cDNA synthesis kit for mRNA detection, or with Takara™ microRNA transcription kit for microRNA detection. Real time PCR was carried out on Bio-Rad S1000 PCR instrument and each sample was analyzed in triplicate. PCR data were normalized to GAPDH or U6 snRNA expression for mRNA and miRNA respectively.
Primers for miR-125b and U6 were obtained from GeneCopoeia (GuangZhou Ribobio Co.Ltd, China), The antisense primer for miRNAs was bought from Takara (Dalian, China). Gab2 primers as follows: sense, 5’-CGC TGC TAGAC AAC AGC CGA CTT CAC C-3’ and antisense, 5’-GCC CAC AAT CAT TTT CCC T -3’.
Primers for miR-125b and U6 were obtained from GeneCopoeia (GuangZhou Ribobio Co.Ltd, China), The antisense primer for miRNAs was bought from Takara (Dalian, China). Gab2 primers as follows: sense, 5’-CGC TGC TAGAC AAC AGC CGA CTT CAC C-3’ and antisense, 5’-GCC CAC AAT CAT TTT CCC T -3’.
4
Chondrocyte RNA Extraction and qPCR Analysis
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The cartilaginous tissues or primary cultured chondrocytes from mice were collected and lysed in TRIzol (Invitrogen) for RNA isolation according to the manufacturer’s standard protocol. cDNA was synthesized from 1 μg RNA Maxima First Strand cDNA Synthesis kit (Takara). Real-time PCR was performed on ABI Fast7500 with Maxima SYBR Green qPCR Master Mix (Takara). The primer pairs have been previously described25 (link),55 (link)–58 (link) and are included in Supplementary Table 3 . Fluorescence qPCR was performed by real-time fluorescence qPCR instrument (qTOWER3G, Jena Bioscience) and its application software (qPCRsoft 3.4). Real-time PCR results were analysed by Microsoft Excel (2306 Build 16.0.16529.20164).
5
Quantitative Analysis of Gene Expression
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Total mRNA was obtained from clinical fresh tissues or cultured cells using QIAZOL (Qiagen, Shanghai, China). And 1 μg extracted mRNA was used for Complementary DNA (cDNA) synthesis with random primers and Maxima First Strand cDNA Synthesis Kit (Takara Bio, Inc., Otsu, Japan). Real-time quantitative PCR (RT-qPCR) was performed according to the manufacturer’s instructions using the SYBR Green Master Mix (TAKARA) and LightCycler480II system (Roche). GAPDH used as the reference gene for NAP1L1 or HDGF or c-Jun; and the relative expression of RNAs was calculated using formula [30 (link)]. The primers were listed in the Supplementary Table 3 .
6
RNA Extraction and Quantitative PCR
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Cells were lysed and RNA was extracted from them 36 h after transfection using the QIAZOL reagent (Qiagen, Shanghai, China). Complementary DNA (cDNA) was then synthesized from 1 µg extracted RNA using random primers and the Maxima First Strand cDNA Synthesis Kit (Takara Bio, Inc., Otsu, Japan) in the eBio-Rad CFX 96 system. The SYBR®Green Master Mix was used to perform quantitative real-time PCR in the Bio-Rad T100 detection system. All primer sequences are presented in Supplementary Table 2 .
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