Kit de synthèse de l’ADNc premier brin Maxima pour la RT-qPCR

It is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning. RNase H degrades RNA from RNA-DNA duplexes, which can result in truncated cDNA during reverse transcription of long mRNA. We also recommended using RNase H-minus RTs for template-independent addition of C nucleotides.

cDNA synthesis at higher temperatures ensures successful transcription of RNA with high levels of secondary structure, reducing issues of primer access to template. Therefore, we do recommend to use RT enzymes with high thermostability, e.g. Maxima and Maxima H Minus Reverse Transcriptases, which provide higher yields of full-length cDNA, better sensitivity, and successful transcription of GC-rich templates.

Trace amounts of reagents used in RNA purification protocols may remain in solution and inhibit first-strand synthesis, e.g., SDS, EDTA, guanidine salts, phosphate, pyrophosphate, polyamines, spermidine. To remove trace contaminants, we recommend re-precipitating the RNA with ethanol and washing the pellet with 75% ethanol, or re-purifying the RNA.

RNA purity and integrity are essential for synthesis and quantification of cDNA. Always assess the integrity of RNA prior to cDNA synthesis. Use freshly prepared RNA. Multiple freeze/thaw cycles of the RNA sample and synthesized cDNA is not recommended. Avoid RNase contamination and discard low quality RNA.

It is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning. RNase H degrades RNA from RNA-DNA duplexes, which can result in truncated cDNA during reverse transcription of long mRNA. We also recommended using RNase H Minus RTs for template-independent addition of C nucleotides. In contrast, reverse transcriptases with intrinsic RNase H activity are often favored in 1-step RT-qPCR applications.