First-strand reaction protocol
General precaution against RNase degradation of template RNA should be taken when setting up first-strand reactions. Such precautions include use of nuclease-free water, RNase inhibitor, RNase-free tubes and sterile pipet tips with filters. The following procedure can be used as a guideline for preparing a 20 µl first-strand cDNA reaction.
Materials not included:
• Sterile, nuclease-free water
• Primer (oligo dT(15-20) or random hexamers or gene-specific)
• dNTP mix (dATP, dCTP, dGTP, dTTP, cat. no. N2050L)
• RNA template
• RNase Inhibitor (cat. no. Y9240L)
• RNase H (cat. no. Y9240L)
1. Add the following components to an RNase-free microcentrifuge tube on ice. For more than one reaction, prepare a master mix.
|50 µM oligo dT(15–20) or
50 µM random hexamers or
10 µM gene-specific primer
|1 to 2 µl
|5 mM dNTP mix
|RNA template: 1 ng to 1 µg total RNA or 1 to 250 ng mRNA
|Sterile, nuclease-free water
||To total volume of 12 µl
2. Heat microcentrifuge tube to 65°C for 5 minutes and quickly cool on ice for 2 minutes to anneal primer to RNA template. Spin tube briefly to collect condensate.
3. Add the following components (to each first-strand reaction) to the microcentrifuge tube on ice:
|5X M-MLV Reverse Transcriptase RNase H- Buffer
|100 mM DTT
|RNase Inhibitor (optional) or
|200 U EnzScript Reverse Transcriptase
4. Incubate reactions as follows:
• 25°C for 2 minutes (oligo dT(15–20), gene-specific primer) or 25°C for 10 minutes (random hexamer)
• 42°C for 30–60 minutes
• Heat kill at 70°C for 15 minutes
5. Use cDNA in downstream application or store at –20°C. For RT-PCR, 1–2 µl cDNA from the first-strand reaction is typically added as template to PCR.
Optional: Remove RNA strand prior to PCR by adding 1 µl RNase H (cat. no. Y9220L) to cDNA:RNA hybrid, incubate at 37°C for 20 minutes and 65°C for 10 minutes (heat kill). RNase H treatment is recommended for amplification of long amplicons (>5 kB).
QUALITY CONTROL ANALYSIS
Unit activity is measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X M-MuLV RT RNase H– Buffer and added to 50 µl reactions containing 20 µg/ml poly r(A) RNA, oligo (dT) DNA, 1X RT Buffer, 3H-dTTP and 250 µM dTTP. Reactions were incubated 10 minutes at 37°C, placed on ice and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, A8.25–A8.26).
Reverse transcriptase function is determined by the enzyme’s ability to generate a 9.4 kB cDNA transcript. Following two-step RT-PCR, the 9.4 kb amplicon was visualized by agarose gel electrophoresis.
Protein concentration (OD280) is determined by OD280 absorbance.
Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample.
Single-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.
Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.
Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.
E. coli 16S rDNA contamination is evaluated using 5 µl replicate samples of enzyme solution denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.
Non-specific RNase contamination is assessed using the RNase Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.
FREQUENTLY ASKED QUESTIONS AND TROUBLESHOOTING
For Frequently Asked Questions (FAQ) and troubleshooting please click here .