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Enzymes for Molecular Biology

RNA Analysis

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:

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Reverse transcriptases

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

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.

RNA digestion and protection

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.

* At low salt concentrations (0 to 100 mM NaCl), RNase A cleaves single-stranded and double-stranded RNA as well the RNA strand in RNA-DNA hybrids. At NaCl concentrations of 0.3 M or higher, RNase A specifically cleaves single-stranded RNA.

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FAQs about RNA analysis

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