Induro Reverse Transcriptase is a group II intron-encoded RT that exhibits high processivity, increased thermostability, and increased tolerance of inhibitors in the synthesis of cDNA from RNA. It is an ideal enzyme for challenging cDNA synthesis from long transcripts, RNAs with strong secondary structures, and RNA samples with inhibitors. With improved 5' sequencing coverage of long transcripts, Induro Reverse Transcriptase can enable RNA-seq applications such as direct RNA and long read cDNA sequencing workflows.
Induro Reverse Transcriptase can synthesize a full-length 12 kb cDNA product within 5 min. at 55°C. In vitro transcribed poly(A) RNA templates (1 kb, 4 kb, 8 kb or 12 kb) were used to investigate full-length cDNA synthesis. After first-strand cDNA synthesis, RNA was hydrolyzed immediately by NaOH. Subsequently, an aliquot of the cDNA products was used to make full-length ds cDNA in the presence of a 5´ specific primer. Equal volume of ds cDNA was analyzed on an agarose gel.
Figure 2: Thermostability of Induro Reverse Transcriptase allows robust synthesis at higher reaction temperatures
Induro Reverse Transcriptase can make a full-length 12 kb cDNA product in 10 min. using incubation temperatures of 45°C to 60°C. In vitro transcribed poly(A) RNA templates (1 kb, 4 kb, 8 kb or 12 kb) were used to investigate full-length cDNA synthesis. After first strand cDNA synthesis, RNA was hydrolyzed immediately by NaOH. Subsequently, an aliquot of the cDNA products was used to make full-length ds cDNA in the presence of a 5´ specific primer. Equal volume of ds cDNA was analyzed on an agarose gel.
Figure 3: Induro Reverse Transcriptase generates highest yields of long cDNA
Induro Reverse Transcriptase generates the highest product yields for cDNA ≥ 8 kb. RNA templates were in vitro transcribed poly(A) RNA (1 kb, 4 kb, 8 kb, 10 kb or 12 kb). After first strand cDNA synthesis, RNA was degraded and the second strand cDNA synthesis was performed in the presence of a 5´ specific primer.
Figure 4: Induro Reverse Transcriptase enables long cDNA synthesis from complex human RNA
Induro Reverse Transcriptase can synthesize products greater than 14 kb from human total RNA using a 10-minute incubation at 50°C, 55°C or 60°C. After first strand cDNA synthesis, an aliquot was amplified by PCR using LongAmp® Taq 2X Master Mix (NEB #M0287).
Figure 5: Induro Reverse Transcriptase is more inhibitor-resistant than TGIRT
Induro Reverse Transcriptase (I) shows a higher tolerance to inhibitors commonly present in RNA biological samples than TGIRT®-III Enzyme (T). 1 μg of in vitro transcribed 8 kb poly(A) RNA was used in a 20 μl cDNA synthesis reaction with oligo dT23VN. All reaction components including the RT enzymes were mixed well on ice before the inhibitor was added. The cDNA synthesis condition for Induro was 55°C, 10 min. followed by 95°C, 1 minute. The cDNA synthesis condition for TGIRT was 60°C, 60 min. followed by 95°C, 1 min. The amounts of inhibitors were the final concentration in the first strand cDNA synthesis. The second-strand cDNA synthesis was performed in the presence of a 5´ specific primer.
Libraries of cDNA were prepared using either Induro RT or Maxima™ RT from total mouse brain RNA before sequencing on an Oxford Nanopore® GridION®. 3.25M reads were sampled from each library (seqtk sample) and aligned to the GRCm39 transcriptome (minimap 2.17). Average 5′→3′ coverage was plotted for each transcript length group with the top 50% of transcripts shown in a heatmap (yellow = high coverage). Reads mapping with mapQ ≥ 1 to either a 3.5 kb transcript (pelkhb2) (A), or a 8.2 kb transcript (adgrl1) (B) are shown in IGV (2.14.0) with Induro and Maxima RT. Induro libraries contain longer transcripts across all transcript length groups.
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