Enzymes to analyze RNA | Reverse transcriptases and RNA ligases

All things RNA: amplify, sequence, ligate, label

There is a suite of enzymes available to manipulate RNA for downstream analysis of gene expression. Reverse transcriptases generate complementary DNA (cDNA) from a transcript. The cDNA can be labeled or amplified for cloning or quantitative studies. RNA ligases join bases, labels and adapters, RNA polymerases add ribonucleotides, and ribonucleases remove them.

Detect and manipulate RNA through amplification

Reverse transcriptases are enzymes able to polymerize a strand of DNA (cDNA) that is complementary to an original RNA template. RNA is susceptible to degradation by RNases and using a reverse transcriptase enzyme to produce cDNA overcomes the problems of working with mRNA. The cDNA becomes the stable template in a variety of downstream applications for RNA studies such as the analysis of gene expression. Regular PCR, qPCR, one-step RT-qPCR or isothermal methods can be used for amplification of the cDNA template. Amplification can be followed by cloning with conventional enzyme protocols or by ligation-independent cloning utilizing the 3’→5’ exonuclease activity of T4 DNA polymerase.

All reverse transcriptase enzymes (RNA-dependent DNA polymerases) can be used to:

Choose a reverse transcriptase enzyme or kit according to the requirements of your RNA template or application.

	RNase H (+/-)	Specialized applications
P7600L EnzScript	Minus	For long cDNAs and libraries with a high percentage of full-length cDNA; applications requiring template switching, i.e., RNA-Seq
P7720L StableScript	Minus	For long cDNAs; improved processivity, inhibitor resistance and thermostability (optimal temperature 55°C)
205111 Omniscript Kit	Plus	Labeling for microarrays; specially designed for reverse transcription with any amount of RNA; optimized buffer; no RNase H step needed
RT32 Reverse Transcriptase	Minus	For full-length cDNAs up to 7 kb; starting material 10 pg–5 µg total RNA or 10 pg–500 ng mRNA; for amplification of difficult RNA templates (optimal temperature 50°C)
RT31 Reverse Transcription Kit	Minus	Kit includes reverse transcriptase (RT32) and RNase inhibitor, 2x Master Mix and RNase H (for optional step)

RNA polymerases and ligases

RNA polymerases, or more specifically DNA-directed RNA polymerases, are enzymes that synthesize RNA from a DNA template. The enzyme Poly(A) polymerase uses single-stranded RNA as a primer to add a poly(A) tail to RNA by catalyzing the incorporation of adenine residues into the 3’ termini of RNA.

T4 RNA ligases are useful enzymes for RNA analysis particularly upstream of procedures such as high-throughput RNA sequencing and microarrays. T4 RNA ligases 1 and 2 are enzymes that can label, circularize or perform intermolecular ligation of RNA by joining adjacent 3'-OH and 5'-PO₄ polynucleotides. Attachment of adapters to RNA 3'-ends with T4 RNA ligase 1 is a useful first step for RNA quantification and discovery by RT-PCR and high-throughput sequencing.

Manipulating RNA is made easy with these polymerases and ligases.

	Action	Applications
P7180L T7 RNA Polymerase Coming soon	Synthesizes RNA in 5’→ 3’ direction off DNA template; specific for the T7 promoter	In vitro mRNA synthesis Labeling of RNA probes RNA generation for RNA- studies Expression control via anti-sense RNA Synthesis of sgRNA
P7460L Poly(A) Polymerase	Catalyzes addition of AMP from ATP to 3’-OH of RNA for poly(A) tailed RNA	3'-labeling of RNA with ATP or Cordycepin Poly(A) tailing of RNA Poly(A) tail enhances translation of RNA transferred into eukaryotic cells
L6050L T4 RNA Ligase 1 Coming soon	Single-stranded RNA ligase; also joins single-stranded DNA molecules	Ligation of single-stranded RNA or DNA 3'-end labeling of single stranded RNA or DNA
L6080L T4 RNA Ligase 2 Coming soon	Ligates nicks on dsRNA; can ligate the 3’-OH of RNA to the 5’-PO₄ of DNA in double stranded hybrids	Templated joining of multiple oligonucleotides Synthesis of RNAs containing site-specific modifications or labels Nick ligation of dsRNA Ligation of 3’-OH of RNA to 5’-PO₄ of DNA in a DNA/RNA duplex
L6070L T4 RNA Ligase 2 (Truncated) Coming soon	Catalyzes phosphodiester bond formation between a pre-adenylated 5’ phosphate (DNA or RNA) and the 3’ hydroxyl of RNA	Linker ligations with pre-adenylated 5’ DNA to 3’ hydroxyl RNA Cloning and sequencing small RNAs

Digesting RNA and protecting RNA

Ribonuclease protection assay (RPA) with the enzyme RNase A is a technique used to determine relative or absolute transcript abundance and to map mRNA termini and intron/exon boundaries.

Single base substitutions can be detected and localized by a simple enzyme method that involves RNase A cleavage of single base mismatches in RNA:DNA heteroduplexes.

Digest RNA with one of these specific endoribonucleases. Protect your RNA with one of our RNase Inhibitors.

	Action	Applications
RP145, 19101 RNase A	Endoribonuclease that degrades ssRNA at C and U residues*	Determining relative or absolute transcript abundance Mapping mRNA termini and intron/exon boundaries (RPA) Mapping single-base mutations in DNA or RNA Removal of RNA from plasmid and genomic DNA preparations Removal of RNA from recombinant protein preparations
Y9220L RNase H (Enzymatics)	Endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA hybridized to DNA	RNase H activity degrades the RNA template of a DNA:RNA complex releasing single-stranded cDNA as a template for synthesis of the second-strand cDNA
RT34 RNase H (BLIRT)	Endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA hybridized to DNA	Key enzyme in the removal of mRNA after first-strand cDNA synthesis Treatment of cDNA with RNase H prior to PCR improves sensitivity Removal of poly(A) tails on mRNAs after hybridization with oligo(dT) Site-specific enzymatic cleavage of RNA
Y9240L RNase Inhibitor	Porcine-derived non-competitive inhibitor of RNase A, B, C; does not inhibit RNase H activity.	Protects RNA from degradation; commonly used as a precautionary measure in enzymatic manipulations of RNA
RT35 RNase Inhibitor Hu (human)	Human placental protein inhibits ribonuclease (RNase) activity of common eukaryotic enzymes such as RNase A, RNase B, RNase C	RNA-related molecular diagnostics RNA isolation and purification cDNA synthesis, RT-PCR, RT-qPCR in vitro transcription and translation RNase-free monoclonal antibody preparation

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