Collection: Reverse Transcription & cDNA Synthesis

Oligo(dT) anneals to the poly(A) tail, so only mRNA is copied — clean, but biased toward the 3' end and dependent on intact RNA. Random hexamers prime anywhere and therefore capture degraded, non-polyadenylated and prokaryotic RNA, at the cost of shorter and less proportional products. Gene-specific primers give maximum specificity for a single target. Many kits deliberately blend oligo(dT) with random primers to get coverage and yield together.

Reactions commonly take between about 100 ng and 1 µg of total RNA in a 20 µl volume, adjusted to target abundance and how precious the sample is. Store cDNA at −20 °C, either neat or in 10 mM Tris-HCl pH 8.0 with 0.1 mM EDTA. For qPCR, dilute cDNA made from more than 1 µg of input at least tenfold, and keep the cDNA volume below 10% of the qPCR reaction volume.

Wild-type MMLV and AMV reverse transcriptases carry RNase H activity that degrades the RNA strand of the RNA:DNA hybrid while synthesis is still running, truncating long products. Engineered RNase H-minus enzymes have that activity greatly reduced, which improves full-length yield from long transcripts and allows higher reaction temperatures that melt out secondary structure.

Template switching was designed for exactly this. When PrimeScript reaches the 5' end of the RNA it adds a short stretch of non-templated nucleotides to the cDNA; a SMARTer oligo base-pairs with that stretch and extends the template, so the enzyme keeps copying to the end of the oligo and marks the true 5' terminus. The result is full-length, ligation-free cDNA from low-input material, and it underpins the SMARTer kits used for RNA-seq library preparation.

Danyel Biotech is the official Israeli distributor for Takara Bio and holds the PrimeScript and SMARTer reverse transcription range locally, with cold-chain shipping and application advice on primer choice and RNA input. Request a quote from this page or send the catalog numbers you already use.