Total RNA from human chondrocytes and rat chondrocytes was extracted using TRIzol®. The RNA was reverse transcribed to cDNA by reverse transcription using an mRNA reverse transcription kit (CW2569, Cwbio) and miRNA reverse transcription kit (CW2141, Cwbio). Primers were designed and synthesized by Beijing Tsingke Biotechnology, and the sequences are listed in Table 1 . Real-time Quantitative PCR (RT-qPCR) was performed using fluorescence quantitative PCR kit (PIKOREAL96, Thermo). The fluorescence intensity during the reaction was recorded in real time. The gene expression was calculated by 2−ΔΔCt method compared with β-actin, U6, or 5S.
Mirna reverse transcription kit
Manufactured by CWBIO
Sourced in China
The MiRNA reverse transcription kit is a laboratory tool designed to facilitate the reverse transcription of microRNA (miRNA) molecules. This kit enables the conversion of miRNA into complementary DNA (cDNA) for subsequent analysis and applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Cw2569, by CWBIO (4 mentions)
Mrna reverse transcription kit, by CWBIO (3 mentions)
Ultrasybr mixture, by CWBIO (2 mentions)
Pikoreal96, by Thermo Fisher Scientific (1 mentions)
Cw2141, by CWBIO (1 mentions)
Ultrasybr mixture, by Thermo Fisher Scientific (1 mentions)
4 protocols using mirna reverse transcription kit
1
Quantitative Analysis of Gene Expression
2
Quantitative Gene Expression Analysis
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Total RNA was extracted from cells or tumor tissues using TRIzol reagent (15596026, Thermo). cDNA was synthesized using an mRNA reverse transcription kit (CW2569, CWBIO) and miRNA reverse transcription kit (CW2141, CWBIO). The expression levels of target genes were analyzed by UltraSYBR Mixture (CW2601, CWBIO) and the 2−ΔΔCT method. Primer sequences are shown in Table 1 .
3
Quantifying LOXL1-AS1, miR-761, and PTEN Levels
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LOXL1-AS1, miR-761, and PTEN levels in cells and tissues were measured using qRT-PCR. Briefly, total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality and quantity were assessed with agarose gel electrophoresis and A260/A280 ratio with spectrophotometer. The A260/A280 ratios for all RNA preparations were about 1.8–2.0. For miRNA, a miRNA reverse transcription kit (CW2141; CWBIO, Beijing. China) was used to synthesize cDNA. For mRNA, an mRNA reverse transcription kit (CW2569; CWBIO) was applied. Ultra SYBR Mixture (Thermo Fisher Scientific) was applied to test the gene level, which was calculated using the 2−ΔΔCt method. The reaction parameters set by the PCR instrument as follows: 95°C for 10 minutes followed by 45 cycles consisting of 95°C for 15 seconds, 58°C for 30 seconds and 68°C for 60 seconds. U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the internal reference for miRNA and mRNA, respectively. Primer sequences are shown in Table 1 .
4
RNA Extraction and Gene Expression Analysis
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TRIzol reagent (15,596,026, Thermofisher, Waltham, MA, USA) was applied to extract RNA from samples. cDNA was synthesized using an mRNA reverse transcription kit (CW2569, CWBIO, Beijing, China) and miRNA reverse transcription kit (CW2141, CWBIO, Beijing, China). The sequences of target genes were searched on NCBI, and primer5 software was performed to design primers. The expression levels of target genes were analyzed by UltraSYBR Mixture (CW2601, CWBIO, Beijing, China) and 2−ΔΔCT. Primer sequences are shown in Table 1 .
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