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
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.

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
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
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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