Total RNA was extracted from cells using RNeasy Kits, according to the protocol provided by the manufacturer (QIAGEN, Hilden, Germany). The superscript III first‐strand synthesis kit (TaKaRa) was used to synthesize complementary DNA (cDNA) from total RNA. Then, RT-qPCR was performed on the ABI Prism 7900 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA) using a SYBR Green RT-PCR kit (TaKaRa). Expression levels were normalized to expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Superscript 3 first strand synthesis kit
Manufactured by Takara Bio
Sourced in China, Japan
The Superscript III First-Strand Synthesis Kit is a reagent system for the reverse transcription of RNA to cDNA. It contains the Superscript III reverse transcriptase enzyme, reaction buffer, dNTPs, and other necessary components for the first-strand cDNA synthesis process.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 7900 sequence detection system, by Thermo Fisher Scientific (5 mentions)
Sybr green rt pcr kit, by Takara Bio (5 mentions)
Rneasy kit, by Qiagen (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Sybr green probe, by Takara Bio (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
6 protocols using superscript 3 first strand synthesis kit
1
RNA Extraction and RT-qPCR Analysis Protocol
2
Quantifying Tendon Gene Expression
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The mRNA expression levels of tendon‐related genes were determined using qPCR. Total RNA was extracted from cells using TRIzol reagent, according to the protocol provided by the manufacturer (Takara, Dalian, China). cDNA was synthesized from total RNA using a Superscript III First‐Strand Synthesis Kit (TaKaRa). qPCR was performed using a SYBR Green RT‐PCR kit (TaKaRa) and an ABI Prism 7900 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA). Expression levels were calculated relative to the expression of the housekeeping gene glyceraldehyde 3‐phosphate dehydrogenase (GAPDH).
3
Quantifying Tendon-Related Gene Expression
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The mRNA expression levels of tendon-related genes of collagen type I alpha china (COL1A1), tenascin-C (TNC), tenomodulin (TNMD), and scleraxis (SCX) were determined using real-time quantitative polymerase chain reaction (RT-qPCR). One microgram of total RNA was extracted from TSPCs using TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer's protocol, and then 1 μg of RNA was converted to complementary DNA (cDNA) using a Superscript III First-Strand Synthesis Kit (TaKaRa). qPCR was performed using a SYBR Green RT-PCR kit (TaKaRa) and an ABI Prism 7900 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to calculate the relative expression level of the target gene. The PCR primer sequences are shown in Table 1 .
4
Quantifying Tendon Gene Expression
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The original five pairs and another five pairs of tendons were used for verification. The samples were harvested and analyzed as described above. cDNA was generated from the total RNA using the Superscript III First-Strand Synthesis Kit (TaKaRa) according to the manufacturer’s instructions. qRT-PCR was conducted using the SYBR Green RT-PCR Kit (TaKaRa) and ABI Prism 7900 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA). The primers are listed in Table 2 . mRNA and lncRNA expression levels were normalized to GAPDH expression. The relative changes in gene expression were calculated using the 2–∆∆Ct method (16 (link)). The analysis was conducted three times independently.
5
Quantifying Adipogenesis-Related Gene Expression
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The mRNA expression levels of adipogenesis related gene were determined by qRT‐PCR. TRIzol regent was used to extract total RNA from cells, according to the protocol provided by the manufacturer (Takara). Superscript III first‐strand synthesis kit (TaKaRa) was used to synthesize cDNA from total RNA. SYBR Green RT‐PCR kit (TaKaRa) and an ABI Prism 7900 Sequence Detection System (PE Applied Biosystems) were used to perform qPCR. Expression of the housekeeping gene GAPDH was used as relative expression control.
6
Quantitative PCR Analysis of GAB1
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Total RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). cDNA was synthesized using the SuperScript III First-Strand Synthesis kit following the manufacturer's protocol (TaKaRa, Shiga, Japan). Quantitative polymerase chain reaction (PCR) targeting GAB1 was performed in a 20 μL reaction containing 1 μg of cDNA template, 1 μL of SYBR Green probe (TaKaRa). The PCR reactions were performed on a ViiA 7 Real-Time PCR System (Applied Biosystems, Waltham, Massachusetts, USA) with the following condition: 50°C, 2 min; 95°C, 10 min; 40 cycles of 95°C, 15 s and 60°C, 1 min. Samples were run in triplicate concurrently with a 10× serial dilution of sample cDNA as a standard.
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