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Enzymes for Molecular Biology

RNA Analysis

Amplify and sequence, ligate and 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 complimentary 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:

    RNase H (+/-)
 Specialized applications
P7600L
EnzScript		 Coming soon  Minus For long cDNAs and libraries with a high percentage of full-length cDNA;
applications requiring template switching, i.e., RNA-Seq
P7720L
StableScript		 Coming soon

Minus

For long cDNAs; improved processivity, inhibitor resistance and thermostability
(optimal temperature 55°C)
205111
Omniscript Kit		 Available  Plus Labeling for microarrays; specially designed for reverse transcription
with any amount of RNA; optimized buffer; no RNase H step needed

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.

    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		 Coming soon

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.

    Action Applications
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		 Coming soon Endoribonuclease that 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
Y9240L
RNase Inhibitor		 Coming soon 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

FAQs about RNA analysis

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