Reverse-iT™ 1st Strand Synthesis Kit From ABgene

My research involves expression analysis of many different genes, so I have tried several different kits for first-strand cDNA synthesis and have found the Reverse-iT™ 1st Strand Synthesis Kit very satisfactory. The kit supplies all of the reagents needed to generate high yields of full-length cDNA. The kit includes a unique (patented) reverse transcriptase (RT) that provides good dynamic range, temperature stability and enhanced sensitivity. RNases inhibitor is included in the enzyme mix. The kit also provides anchored oligo-dT primers and random decamers for first strand synthesis, positive control template and primers. Two buffers are provided: for one-step and two-step protocols.

The manufacturer recommends transcribing from up to 1 µg of total RNA or 10-100 ng of mRNA. For my experiments, I used total RNA for quantative real-time PCR and found that a range of 500 ng to 1 µg gave me satisfactory results with both the housekeeping gene: GAPDH, and experimental genes. The reaction set up consists of two steps. First, mix your RNA template, double-distilled sterile water, and random decamers or anchored oligo-dT. The company advises the use of anchored oligo-dT. These primers have been designed to anneal at the mRNA/poly-A junction rather than at a random point within the poly-A tail. By eliminating transcription through the poly-A tail, use of this primer provides more effective cDNA synthesis. The use of anchored oligo dT primer often results in increased cDNA yield and specificity of PCR products. However, using the anchored oligo-dT primer alone will produce less cDNA when the mRNA/poly-A junction is missing; this can happen during total RNA preparation. In addition, in some species, RNA does not have a polyA tail. I found that I obtain better results when I use mix of anchored oligo-dT primers and random decamers.

The next step is incubation at 70°C for 5 minutes. I have found that incubation at such a high temperature may sometimes cause degradation of the sample if the total RNA sample contains Ca²⁺ or Mg²⁺ ions from previous DNase treatment. So, I decreased the incubation temperature to 65°C and still got good results. During incubation, you need to prepare the reaction mix from the reaction buffer, dNTPs, DTT and Reverse-iT™ enzyme blend and then mix it with the heated RNA sample.

Then you incubate the reactions for about one hour at 42-57°C. I do all my incubations routinely at 42°C for one hour. I have found, especially with rarely transcribed genes, that adding another 0.3 µl of enzyme blend after 45 minutes of incubation greatly improves transcription efficiency.

In conclusion, The Reverse-iT™ 1st Strand Synthesis Kit is a convenient, reliable, easy to use product and gives excellent reproducibility. I highly recommend it.