To validate the RNA-seq results, quantitative real-time PCR (qRT-PCR) was performed in this study. RNA was extracted using the HiPure Total RNA Mini Kit (Magen, Guangzhou, China). Later, RNA was reverse transcribed to cDNA using the HiScript II 1st Stand cDNA Synthesis Kit (+ gDNA wiper; Vazyme, Nanjing, China). The qRT-PCR was performed in triplicate for each sample using the 2 × RealStar Green Fast Mixture (with ROX) as per the manufacturer’s instruction manual. The qRT-PCR amplification was performed using the StepOne Real-Time PCR Instrument (Applied Biosystems, Thermo Fisher, USA) and corresponding software (Applied Biosystems). The tomato Actin (Solyc03g078400) gene was used as the internal control99 (link). The relative expression levels of the genes from three biological replicates were calculated using the 2−△△Ct method100 (link). All primers for qRT-PCR are listed in Supplementary Table S1 .
Hiscript 2 1st stand cdna synthesis kit
Manufactured by Vazyme
Sourced in China
The HiScript II 1st Strand cDNA Synthesis Kit is a laboratory tool used for the conversion of RNA into complementary DNA (cDNA). It contains the necessary reagents and enzymes required for this process.
Automatically generated - may contain errors
2 protocols using hiscript 2 1st stand cdna synthesis kit
1
Validating RNA-seq Using qRT-PCR
2
Hornet Venom cDNA Library Construction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA libraries of these hornet venoms were built. We used the HiS-cript®II 1st Stand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd) to reverse transcribe RNA into first-strand cDNA. Double-strand cDNA was then synthesized by utilizing first-strand cDNA, a 5 ′ PCR primer: 5 ′ -AAGCAGTGGTATCAACGCAGAGT-3 ′ and the 3 ′ SMARTTM CDS III/3 ′ PCR Primer: 5 ′ -ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3' (N = A, G, C, or T; N-1 = A, G, or C) in the second-strand to allow long distance PCR. Finally, this cDNA was used as a template with a 5 ′ primer (5 ′ -ATGAGTGCCGAAGCTTTAGCT-3 ′ ) and the 3 ′ SMART™ CDS III/3 ′ PCR Primer for PCR. We used the following settings: 1 min at 94 • C, 30 cycles for 10 s at 92 • C, 30 s at 53 • C, and 40 s at 72 • C, followed by a final 10 min at 72 • C. We used a general rTaq enzyme (TaKaRa Biotechnology Co.Ltd., Dalian, China).
The resulting PCR amplification products were then purified with a Fast Gel DNA Extraction Mini Kit (Vazyme Biotech Co., Ltd). The purified cDNA was then linked to a pMD™19-T vector (TaKaRa Biotechnology Co. Ltd.), and the ligation products were inserted into incubated DH5a competent cells for cloning and sent to TsingKe Biotechnology Co. Ltd. (China) for cDNA sequencing.
The resulting PCR amplification products were then purified with a Fast Gel DNA Extraction Mini Kit (Vazyme Biotech Co., Ltd). The purified cDNA was then linked to a pMD™19-T vector (TaKaRa Biotechnology Co. Ltd.), and the ligation products were inserted into incubated DH5a competent cells for cloning and sent to TsingKe Biotechnology Co. Ltd. (China) for cDNA sequencing.
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!