Reverse Transcription Protocol

Protocol for reverse transcription using ReadyScript cDNA Synthesis (RDRT) Reagent

Place components on ice. Mix and then briefly centrifuge to collect contents at the bottom of the tube

Reagent	Volume for 20 μL reaction	Final Concentration
ReadyScript cDNA Synthesis Mix (RDRT)	4 μL	1X
RNA template	variable	1 μg to 10 pg total RNA
Rnase/DNase-free water	variable
Total Volume	20 μL

Note: For smaller or larger reaction volumes, components should be scaled proportionately

Reaction

• Combine reagents in 0.2 mL micro-tubes or 96-well plate sitting on ice.
• After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube.
• Incubate:
  • 5 minutes at 25 °C
  • 30 minutes at 42 °C
  • 5 minutes at 85 °C
  • Hold at 4 °C
• After completion of cDNA synthesis, use 1/5th to 1/10th of the first-strand reaction (2-4 μL) for PCR amplification. If desired, cDNA product can be diluted with 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA and stored at -20 °C.

Guidelines for Reverse Transcription-qPCRMinus RT-controls:

Accurate quantification of gene expression by RT-qPCR requires quantitation of genomic DNA contamination in each RNA sample for each gene of interest. The presence of trace amounts of gDNA does not usually interfere with quantification of high copy reference genes, but can have a significant contribution on signal for low copy genes. Even when using primers that are separated by intronic sequence or bridge exon junctions, the presence of genomic DNA can produce positive signals from amplification of pseudogene or off-target PCR product. Therefore, it is important to alwaysinclude the appropriate “no RT” or “minus RT” control reactions in your experimental design.

Since the reverse transcriptase is an integralcomponent of ReadyScript cDNA Synthesis Mix, it is not feasible to construct a formal cDNA synthesis control that includes all components except the RT. The most direct method to test for the presence of genomic DNA is to bypass the RT step and use an equivalent amount of the RNA preparation directly for PCR amplification. For example: if you start with 1 μg of total RNA for cDNA synthesis and use 1/10th of the first-strand reaction as template for qPCR; then use 100 ng of total RNA as template for the minus RT-control qPCR. Any signal from the RNA only reaction is attributable to the presence of genomicDNA.

DNase digestion of total RNA:

If trace levels of genomic DNA obscure accurate quantification of your gene(s) of interest, use a high quality, RNase-free preparation of DNase I to remove residual genomic DNA (DNAse I, Catalog Number AMPD1-1KT). After the DNase digestion, it is essential to remove all traces of DNase activity before proceeding with first-strand synthesis. Suitable RNA purification methods include phenol:chloroform extraction followed by ethanol precipitation, or the use of chaotropic salts and a silica-based RNA purification cartridge or column(GenElute™ Direct mRNA Miniprep Kit, Catalog Number DMN10). Simple “heat-kill” procedures or the use of inactivating slurry solutions are not compatible with ReadyScript cDNA Synthesis Mix.

Related Definitions

cDNA - Complementary DNA (cDNA) is a copy of DNA from a single-stranded RNA or messenger RNA (mRNA) template that is made by the enzyme reverse transcriptase. cDNA can be converted to DNA by DNA polymerase. cDNA, like its template RNA or mRNA, contains only certain fragments of the entire genome and can be useful for studying the expression of specific genes. A cDNA library is a collection of cDNA usually organized by gene.