Total RNA was isolated from HEBCs by using the TRIzol (Invitrogen, Carlsbad, CA, USA). Single-stranded cDNA was synthesized with the PrimeScript Reagent Kit (Promega, USA). Real-time PCR was conducted by using SYBR Premix Ex TaqTM Kit (Applied Biosystems, Foster City, CA, USA). The reaction was run in ABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). GAPDH was used as an endogenous control. Briefly, 2 μL of cDNA was added to 10 μL of the 1×SYBR green PCR master mix with 0.4 μL of Taq polymerase enzyme (RiboBio, China), 0.8 μL of each primer and 6 μL ddH2O to a final volume of 20 μL. The RT-qPCR cycling conditions consisted of: 95° C for 3 min; then 35 cycle amplification for 20 s at 95° C, 30 s at 55° C, 15 s at 72° C; followed by 1min at 72° C. The primers used in this study were synthesized from Sangon Biotech (Shanghai, China). The levels of mRNA were normalized by using the 2-ΔΔCt method.
Primescript reagent kit
Manufactured by Promega
Sourced in United States
The PrimeScript Reagent Kit is a set of reagents designed for reverse transcription and cDNA synthesis from RNA samples. The kit includes enzymes, buffers, and other components necessary for these molecular biology processes. The core function of the PrimeScript Reagent Kit is to facilitate the conversion of RNA into complementary DNA (cDNA) for subsequent analysis or applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (5 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Sybr premix ex taqtm kit, by Thermo Fisher Scientific (3 mentions)
Taq polymerase enzyme, by RiboBio (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Takara Bio (1 mentions)
Sybr premix ex taqtm kit, by Takara Bio (1 mentions)
5 protocols using primescript reagent kit
1
Quantitative Real-Time PCR for Gene Expression
2
Quantitative Analysis of circRNA and miRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from tissues and cells by using the TRIzol (Invitrogen, Carlsbad, CA, USA). Single-stranded cDNA was synthesized with the PrimeScript Reagent Kit (Promega, Madison, USA). The cDNA was carried out starting from 100 ng of total RNA. The relative expression levels of circRNA and miRNA were detected by using the SYBR Green Master Mix (Takara Biotechnology, Dalian, China) in Step One Plus Real-Time PCR system (Applied Biosystem, Foster City, CA, USA). Circ_0000079 was validated via PCR using divergent and convergent primers. Glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control. Briefly, 500 ng of total RNA was reversely transcribed into cDNA with random primers in a total volume of 20 μl. The reactions were initiated in a 96-well optical plate at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s.
3
Quantifying Gene Expression in Tumor Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from tumor tissues by using TRIzol (Invitrogen, Carlsbad, CA, USA). Single-stranded cDNA was synthesized with the PrimeScript Reagent Kit (Promega, USA). Real-time qPCR was conducted by using SYBR Premix Ex TaqTM Kit (Applied Biosystems, Foster City, CA, USA). The reaction was run in ABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). GAPDH was used as an endogenous control. The PCR cycling conditions consisted of: 95 °C for 3 min; then, 35 cycle amplification for 20 s at 95 °C, 30 s at 55 °C, 15 s at 72 °C; followed by 1 min at 72 °C. The primers used in this study were synthesized by Sangon Biotech (Shanghai, China). The expression level was normalized by using the 2−ΔΔCt method.
4
Quantification of miR-26b-5p and KLF10 Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from tissues and cell lines by using the TRIzol (Invitrogen, USA). Single-stranded cDNA was synthesized with the PrimeScript Reagent Kit (Promega, USA). Real-time PCR was conducted by using SYBR Premix Ex TaqTM Kit (TaKaRa). The reaction was run in ABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA). U6 was used as an endogenous control. The RT-qPCR cycling conditions consisted of: 95 C for 10 min; then 35 cycle amplification for 20 s at 95 C, 30 s at 55 C, 15 s at 72 C; followed by 1 min at 72 C. The primers used in this study were synthesized from Sangon Biotech (Wuhai, China). Data were analyzed by using the 2 ÀDDCT method. First, for all test samples and calibration samples, the CT values of target genes were normalized with the CT values of internal reference genes: Calibrator); secondly, normalize the DCT value of the test sample with the DCT value of the calibration sample: DDCT ¼ DCT (test) À DCT (calibrator), and finally, calculate the expression level ratio: 2 ÀDDCT ¼ the ratio of expression amount. 17 The following primers were used: miR-26b-5p (forward 5'-3': ATCTCAGTCC TGTACAGTAC, reverse 5'-3': CGT ACA TGC ACA TAC AGT C); KLF10 (forward 5'-3': ACG GTG CCT CGG GTT GTG; reverse 5'-3': TCG ACC AAT CAG CGG TAA AGG).
5
Analyzing Osteoarthritis Chondrocyte Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from osteoarthritis chondrocytes by using the TRIzol (Invitrogen, Carlsbad, CA, USA)). Single-stranded cDNA was synthesized with the PrimeScript Reagent Kit (Promega, USA). Realtime PCR was conducted by using SYBR Premix Ex TaqTM Kit (Applied Biosystems, Foster City, CA, USA).
The reaction was run in ABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA). GAPDH was used as an endogenous control. The RT PCR cycling conditions consisted of: 95 ˚C for 10 min; then 35 cycle ampli cation for 20 s at 95 ˚C, 30 s at 55 ˚C, 15 s at 72 ˚C; followed by 1min at 72 ˚C. The primers used in this study were synthesized from Sangon Biotech (Shanghai, China). The level of mRNA was normalized to β-actin expression using the 2 -ΔΔCt method.
The reaction was run in ABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA). GAPDH was used as an endogenous control. The RT PCR cycling conditions consisted of: 95 ˚C for 10 min; then 35 cycle ampli cation for 20 s at 95 ˚C, 30 s at 55 ˚C, 15 s at 72 ˚C; followed by 1min at 72 ˚C. The primers used in this study were synthesized from Sangon Biotech (Shanghai, China). The level of mRNA was normalized to β-actin expression using the 2 -ΔΔCt method.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!